Nucleic acid detection and quantification by post-hybridization labeling and universal encoding

ABSTRACT

The present invention provides, among other things, methods and compositions for encoding a substrate for detecting and quantifying target nucleic acids.

RELATED REFERENCES

This application is a division of U.S. non-provisional patentapplication Ser. No. 13/568,884, filed Aug. 7, 2012, which is a 371national phase application of International Application No.PCT/US11/39531, filed Jun. 7, 2011 which claims priority to U.S.provisional patent applications Ser. No. 61/352,018, filed Jun. 7, 2010,Ser. No. 61/365,738, filed Jul. 19, 2010, and Ser. No. 61/387,958, filedSep. 29, 2010, the entire contents of which are herein incorporated byreference.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form ofa text file (entitled “ACM-003US4,” created on Jul. 5, 2016, and 14,971bytes in size) is incorporated herein by reference in its entirety.

BACKGROUND

The multiplexed detection of biomolecules plays an important role inclinical diagnostics, discovery, and basic science. This requires theability to both encode substrates associated with specific biomoleculetargets, and also to associate a detectable signal to the biomoleculetarget being quantified. For multiplexed assays, it is common to usefunctionalized substrates, planar or particle-based, to capture andquantify targets. In the case of particle-based multiplexed assays, eachparticle is functionalized with a probe that captures a specific target,and encoded for identification during analysis. In order to quantify theamount of target captured on a particle, a suitable labeling scheme istypically used to provide a measurable signal associated with thetarget. One class of molecules that is particularly challenging toquantify due to limitations with existing approaches to labeling ismicroRNA (miRNA).

miRNAs are short non-coding RNAs that mediate protein translation andare known to be dysregulated in diseases including diabetes,Alzheimer's, and cancer. With greater stability and predictive valuethan mRNA, this relatively small class of biomolecules has becomeincreasingly important in disease diagnosis and prognosis. However, thesequence homology, wide range of abundance, and common secondarystructures of miRNAs have complicated efforts to develop accurate,unbiased quantification techniques. Applications in the discovery andclinical fields require high-throughput processing, large codinglibraries for multiplexed analysis, and the flexibility to developcustom assays. Microarray approaches provide high sensitivity andmultiplexing capacity, but their low-throughput, complexity, and fixeddesign make them less than ideal for use in a clinical setting.PCR-based strategies suffer from similar throughput issues, requirelengthy optimization for multiplexing, and are only semi-quantitative.Existing bead-based systems provide a high sample throughput (>100samples per day), but with reduced sensitivity, dynamic range, andmultiplexing capacities. Therefore, there is a need for improved methodsfor detecting and quantifying nucleic acids, such as, miRNA.

The multiplexed detection of miRNAs, or any other biomolecules requiresthe ability to encode a substrate associated with each. There are twobroad classes of technologies used for multiplexing—planar arrays andsuspension (particle-based) arrays, both of which haveapplication-specific advantages. While planar arrays rely strictly onpositional encoding, suspension arrays have utilized a great number ofencoding schemes that can be classified as spectrometric, graphical,electronic, or physical.

Spectrometric encoding encompasses any scheme that relies on the use ofspecific wavelengths of light or radiation (including fluorophores,chromophores, photonic structures, or Raman tags) to identify a species.Fluorescence-encoded microbeads can be rapidly processed usingconventional flow-cytometry (or on fiber-optic arrays), making them apopular platform for multiplexing. Most spectrometric encoding methodsrely on the encapsulation of detectable entities for encoding, which canbe very challenging depending on the substrate used. A more robust andgenerally-applicable encoding method is needed to enable rapid,universal encoding of substrates for multiplexed detection.

SUMMARY

The present invention provides improved methods and compositions forhighly efficient, multiplexing, robust and reproducible nucleic aciddetection and quantification. The present invention is, in part, basedon the discovery that a post-hybridization labeling technique can beused with a suitable flow-through scanning or static imaging system forrapid, high-performance nucleic acid detection and/or quantification.Surprisingly, this post-hybridization labeling approach, when used witha versatile particle encoding method, provides scalable multiplexing andattomole sensitivity with a simple workflow. As described in detailbelow, using this robust platform, miRNA expression profiling can beaccurately analyzed for various cancer types within three hours usinglow-input total RNA. Although miRNA was used as an example, inventivemethods and compositions according to the invention may be used todetect any nucleic acids (e.g., DNA, RNA) or other types of analytes.Thus, the present invention represent a significant advance in the fieldof multiplexed biomolecule detection and quantification.

In one aspect, the disclosure in the present application provides asubstrate comprising at least one region bearing one or more nucleicacid probes, each nucleic acid probe comprising a capturing sequence forcapturing sequence for binding a target nucleic acid and an adjacentadapter sequence for binding a universal adapter such that binding ofboth the target nucleic acid and the universal adapter to a same nucleicacid probe is detectable via post-hybridization labeling.

In one aspect, the disclosure in the present application provides anucleic acid probe comprising a capturing sequence for binding a targetnucleic acid and an adjacent adapter sequence for binding a universaladapter such that binding of both the target nucleic acid and theuniversal adapter to the nucleic acid probe is detectable viapost-hybridization labeling.

In one aspect, the disclosure in the present application provides asubstrate comprising one or more universal encoding regions, eachuniversal encoding region bearing one or more single-strandedpolynucleotide templates, wherein each template comprises a stem-loopstructure and a predetermined nucleotide sequence adjacent to thestem-loop structure.

Among other things, the present invention provides a method fordetecting the presence and/or abundance of target nucleic acids in asample. In some embodiments, such a method includes steps of: contactinga plurality of nucleic acid probes with a sample, each nucleic acidprobe comprising a capturing sequence for binding a target nucleic acidand an adjacent adapter sequence for binding a universal adapter;incubating the plurality of probes and the sample, in the presence ofone or more universal adapters, under conditions that permit binding ofboth an individual target nucleic acid and an individual universaladapter to a same individual nucleic acid probe; carrying out a reactionthat allows coupling of the individual universal adapter to theindividual target nucleic acid when hybridized to the same individualnucleic acid probe; and detecting the presence of the one or moreuniversal adapters associated with the plurality of nucleic acid probes,thereby detecting the presence of the target nucleic acids in thesample.

Among other things, the present invention provides a method of encodinga substrate. In some embodiments, such a method includes steps of:providing a substrate comprising one or more encoding regions, eachencoding region bearing one or more single-stranded polynucleotidetemplates; providing a plurality of labeled and unlabeledsingle-stranded encoding adapters, wherein each individualsingle-stranded encoding adapter comprises a sequence designed tospecifically bind an individual polynucleotide template and wherein alabeled single-stranded encoding adapter comprises a detectable moiety;incubating the substrate with the plurality of labeled and unlabeledsingle-stranded encoding adapters under conditions that allow anindividual encoding adapter to bind its corresponding single-strandedpolynucleotide template; and coupling the individual encoding adapter toits corresponding polynucleotide template, thereby encoding thesubstrate.

Also provided is a kit for detecting target nucleic acids. In someembodiments, such a kit includes: a plurality of nucleic acid probes,wherein each individual nucleic acid probe comprises a capturingsequence for binding a target nucleic acid of interest and an adjacentadapter sequence for binding a universal adapter; and one or moreuniversal adapters.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

The drawings are for illustration purposes only, not for limitation.

FIG. 1A, and FIG. 1B illustrate an exemplary schematic for universalencoding and functionalization. In FIG. 1A, hydrogel particles are madeto have several universal encoding regions, each with a stem-loopstructure and unique 4 bp sequence adjacent to the stem-loop, and auniversal anchor in the probe region. In a ligation reaction, encodingadapters are added at varying ratios of fluorescently-modified tounmodified in order to achieve a desired fluorescence level in eachregion while probes are added with linker sequence to add functionalityto the particle probe region. FIG. 1B shows an example of two batches ofparticles with unique code and probes generated using a universalparticle set with varying ligation adapters.

FIG. 2 illustrates an exemplary schematic for universal encoding usingmultiple fluorophores. Multiple adapter variants may be used, each withunique emission spectrum, to encode particles or substrates with morethan one color (or otherwise functional species). The level of eachcolor can be modulated by adjusting the ratio of each adapter variant togive unique signatures of multiple fluorescent colors in the codingregions of the particles.

FIG. 3 illustrates an exemplary schematic of probe-regionfunctionalization using three-species ligation, two-species ligation,and chemical modification.

FIG. 4 illustrates an exemplary schematic showing the use of polymerasewith probe-specific templates to add functionality to particles orsubstrates. Universal anchors (in this case there are two differentanchors) are used with linkers that bear a region specific for oneanchor and a probe sequence. Polymerases are used to extend the anchorsalong the linker, functionalizing the particles/substrates in one ormultiple regions.

FIG. 5 illustrates an exemplary schematic of multi-color scanning with aflow cytometer.

FIG. 6 illustrates an exemplary schematic of single-color scanning witha flow-through device.

FIG. 7 illustrates an exemplary fluorescence scatter plot formultifunctional particles with a single code region functionalized withfour distinct levels of Cy3 (shows in Channel 2) and Cy5 (Channel 4).

FIG. 8A, FIG. 8B and FIG. 8C show an exemplary encoded gel particleassay system. FIG. 8A shows that the workflow of platform includes (i)hybridization of particles with target, (ii) incubation of particleswith universal labeling adapter, ligation enzyme, and fluorescentreporter, and (iii) scanning of particles to determine code identity andamount of target bound. A typical particle includes a fluorescentbarcoded region and a probe-laden region flanked by two inert sections.The central most hole has a fixed value to indicate particleorientation. FIG. 8B show actual PMT fluorescence signatures of 75flow-aligned particles from a 3-s scan segments. FIG. 8C showsmagnified_signatures of individual particles from (b). Overlaid scanswere acquired on different days and demonstrate reproducibility ofanalysis procedure. Scale bar below image is 50 μm.

FIG. 9A, FIG. 9B and FIG. 9C illustrate an exemplary high-throughputflow alignment device and code design. FIG. 9A shows an image of PDMSfocusing chamber attached to glass slide, with inlets and outletattached. Reservoir inlet on the left delivers sheath fluid, whilecentral pipette tip delivers the particle-bearing fluid. Reservoiroutlet on the right serves as a collection point for particles that havebeen scanned. The chamber is mounted on a standard inverted fluorescencemicroscope for scanning runs. FIG. 9B shows images of particles used tooptimize scanner performance. Simple plug particles were scanned tomaximize signal-to-noise ratio (SNR) and frequency response of detectioncircuit. Particles with holes of various areas were used to determinethe minimum differences in size required to distinguish between codinglevels. Scale bars are 50 μm. FIG. 9C illustrates that in the finalparticle design, coding holes were separated by 8 μm, and the lengths ofthe holes were 15, 27.5, and 40 μm for levels 1, 2, and 3, respectively.All holes had a width of 12 μm.

FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D illustrate exemplarypost-hybridization miRNA labeling via ligation to a universal adapter.FIG. 10A shows that DNA probes, lined at their 5′ end throughout theprobe region of encoded hydrogel particles, contain a miRNA _specificsequence adjacent to a universal adapter sequence such that the 3′ endof a captured target would abut the 5′ end of a captured adapteroligonucleotide. The probe is capped with an inverted dT to mitigateincidental ligation and the adapter has a poly(a) spacer to extend itsbiotinylated 3′ end away from the hydrogel backbone for efficientreporting. FIG. 10B shows that after particles are hybridized with totalRNA, T4 DNA streptavidin-phycoerythrin (SA-PE) is used as a fluorescentreporter. FIG. 10C shows that the assay provides about atomole detectionlimits, defined at signal-to-noise=3. FIG. 10D illustrates thatsingle-nucleotide specificity is provided when synthetic let-7a RNA isspiked at 500 amol with particles bearing probes for let-7a, b, c, andd.

FIG. 11 illustrates an exemplary result showing relative ligationefficiency over time. Error bars represent the standard deviation takenover measurements from five particles.

FIG. 12 illustrates an exemplary result showing effect of universaladapter poly(A) tail length on fluorescence signal when usingbiotinylated adapters with a streptavidin-phycoerythrin reporter.Signals are relative to that measured for a tail length of 12 bp.

FIG. 13A, FIG. 13B and FIG. C illustrate an exemplary direct labelingwith fluorophore-conjugated adapters, which are ligated to the end ofcaptured targets. Non-ligated adapters can be rinsed away and theparticles are imaged (or scanned in a flow through device). Fluorescencein the probe-region of the particles is indicative of the amount oftarget present.

FIG. 14 illustrates an exemplary multiplexed detection using multipleadapters with different fluorescent colors. A given probe region of aparticle may contain several unique probes, with common or differingadapters sequences. Adapters bearing fluorophores with unique emissionspectra (fluorescent or other) can be ligated to indicate the capture ofmultiple targets within a given probe region. The amount of fluorescencefrom each fluorophore may be quantified independently to determine theamount of each target present.

FIG. 15A, FIG. 15B and FIG. 15C illustrate an exemplary systemperformance in 12-plex assay. FIG. 15A shows calibration curves forparticle batches, with background-substracted signal plotted againstspiked target amount. miR-210, -221, -222 and let-7a were spiked intothe same incubation mixes at the indicated amounts. The remaining sevennaturally-occurring targets(‘+’ symbols) were spiked into the 27- and243-amol trials to validate performance. For all trials, 200 ng of E.coli total RNA was also spiked in for complexity. Mean COV of targetlevel is 6.35% when considering target levels greater than 5 amol. Eachpoint represents, on average, 19 particles from a single run. FIG. 15Bshows the specificity of let-7a probe in the presence of sequencesclosely related to intended target (see inset box for target set). Weobserved a maximum cross-reactivity of only 27%. FIG. 15C shows cancerprofiling results for four types of human tissue. Error bars representstandard deviation in triplicate measurements on aliquots of the samesingle-patient sample. Amount of total RNA used in assays is 250 ng,unless otherwise noted.

FIG. 16A, FIG. 16B and FIG. 16C illustrate exemplary results showinglimit of detection calculations and calibration curves for neat samples.FIG. 16A shows extrapolation of SNR for determination of limit ofdetection (LOD). The LODs of the four calibration targets (see legend)were calculated by finding the target amount at which the SNR was three.Regression lines with a mean Pearson coefficient of 0.9965 (excludingmiR-222) were used to extrapolate LODs. FIG. 16B shows calibrationcurves for particle batches incubated without spiked E. coli total RNA.Except for the absence of E. coli RNA, conditions are identical to thoseused to construct FIG. 3a . FIG. 16C shows a comparison ofbackground-subtracted signals from neat and E. coli calibrationmeasurements. Clustering of points around the identity line (red)indicates highly specific detection with no noticeable decrease inbinding rates in more complex samples. For all plots, all target levels(except miSpike) have been adjusted for comparison purposes by using thebackground-subtracted signal from the 100-amol miSpike profiles.

FIG. 17 illustrates an exemplary dysregulation classification. A SNR wasused to distinguish dysregulated targets in tissue profiling. The meanand standard deviation of the log-transformed expression ratio werecalculated for each target in each tissue for the triplicate assays. ASNR of three was chosen as the threshold for dysregulation. All 20instances of dysregulation matched observations in the literature

FIG. 18 illustrates exemplary results showing coefficient of variation(COV) of target level as a function of number of particles analyzed. TheCOV of the target level for let-7a in the E. coli calibration scans wasseen to stabilize to a nearly constant value in the 10-15 particlewindow for the five spike-in amounts presented above.

FIG. 19 illustrates a conceptual example of how scanning ofmultifunctional particles could be implemented. Standard cytometeryrecords “events” as instances where the signal from a selected detectorbreaks a threshold, recording single beads as single events, and savingdata for each channel. Multifunctional particles bear functional regionsthat can be doped with triggering entities (that cause scatter forinstance) and single particles are recorded as multiple events. Byanalyzing the shape and time-sequence of these events, and byappropriately designing particles, one can reconstruct from this seriesof events, which ones belong in the same particle.

FIG. 20 illustrates exemplary comparison of scanning fluorescentcalibration beads versus multifunctional particles. Shown is particledesign (top), recorded events at each 1 ms timestamp (middle), anddistribution of events per timestamp for timestamps where at least oneevent was recorded for calibration beads and multi-functional particleswith two fluorescent regions (bottom).

FIG. 21 illustrates exemplary design and use of a standard set of testparticles to assess alignment and consistency of scan.

FIG. 22 illustrates exemplary results of a standard set of testparticles to assess alignment and consistency of scan.

FIG. 23 illustrates exemplary results of nucleic acid detection usingparticles with a single, wide fluorescent region to represent a“barcode” and a narrow probe region.

FIG. 24 illustrates exemplary results of average scans along theparticle length (averaging over half of the width). The bottom signal,second lowest signal, second highest signal, and highest signalcorrespond to 12.5%, 25%, 50%, and 100% fluorescent ligation mixsolutions, respectively.

FIG. 25 illustrates examples of a) particle design and images for eachmixture; and b) average scans over 5 particles for each mixture. (1μm˜3.3 px, numbers in legend represent F1 and F2.)

FIG. 26 illustrates exemplary results of measured fluorescence versusthe adapter amount from each ligation mix.

FIG. 27 illustrates exemplary general particle design for universalencoding.

FIG. 28 illustrates exemplary fluorescent signal obtained in Barcode 1region with varying ratios of fluorescent (Cy3) to non-fluorescentadapter.

FIG. 29 illustrates exemplary plot of events associated with barcodedgel particles. Shown on the left is a plot of YEL fluorescence (used forbarcoding) versus RED2 fluorescence (used for triggering) and on theright YEL fluorescence (used for barcoding) versus GRN fluorescence(used for orientation).

FIG. 30 illustrates exemplary demonstration of 25-plex encoding using 5unique levels of YEL fluorescence on both Barocode 1 and Barcode 2regions of encoded particles. These data have been reconstructed fromraw events saved in a FCS file from the Guava software.

FIG. 31 illustrates exemplary demonstration of attomole sensitivity(left), >3 log dynamic range (left), and single-nucleotide specificity(right) using Firefly BioWorks' custom assay for microRNA targets. Weused Firefly's 3-hour assay (total RNA to results) to detect dilutionsof eleven microRNA targets spiked into 250 ng of E. coli total RNA,reporting the average detector signal versus spike-in amount withinter-run COV (inset). Specificity was assessed by spiking let-7a RNAtarget samples containing particles bearing probes for let-7a, 7b, 7c,and 7d—each which varied by only one or two nucleotides (right).

FIG. 32 illustrates exemplary multiplexed isothermal amplification andcapture assay for panel-based pathogen detection. Fluorescent ampliconsgenerated using reverse transcription helicase-dependent amplification(RT-HDA) will be captured on encoded hydrogel particles in a singlestep. Each particle, bearing probe regions for three signatures of agiven species and porosity-tuned to exclude helicase penetration, willimmediately be scanned in a microdevice without the need for rinsing.The high sensitivity of encoded gel particles and two-levels ofspecificity (amplification and hybridization) will mitigatefalse-positive or negative reads.

FIG. 33 illustrates exemplary proof-of-concept one-pot assays usingstandard PCR and isothermal amplification, demonstrating specificity ofamplification and sensitive detection of ˜11 template copies. Weassessed the specificity of amplification for two targeted regions ofλ-phage DNA, using a one-pot reaction with probes designed against theamplicons generated by two separate primer sets. Template λ-phage wasspiked into (+) samples at ˜11,000 copies for specificity tests, thoughwe were also able to detect amplified product with only ˜11 copiespresent (right). For specificity against human genomic DNA, we spiked˜11,000 copies of human genomic DNA into the reaction with no λ-phagepresent.

FIG. 34 illustrates exemplary amplification primer (left) and ampliconprobe (right) design for multiplexed detection assays. Forward primerswill have a single Cy3 fluorophore. Probes will be designed to have aT_(m) than primers and will be 3′ phosphorylated to avoid incidental3′-extension.

FIG. 35 illustrates exemplary design of barcoded gel particles forspecies-specific amplicon quantification.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

“Adjacent”: As used herein, the term “adjacent” means “next to,”“contiguous,” “adjoining,” “abutting” or having a common boundary.

“Analyte”: As used herein, the term “analyte” broadly refers to anysubstance to be analyzed, detected, measured, or quantified. Examples ofanalytes include, but are not limited to, proteins, peptides, hormones,haptens, antigens, antibodies, receptors, enzymes, nucleic acids,polysaccharides, chemicals, polymers, pathogens, toxins, organic drugs,inorganic drugs, cells, tissues, microorganisms, viruses, bacteria,fungi, algae, parasites, allergens, pollutants, and combinationsthereof.

“Associated”: As used herein, the terms “associated”, “conjugated”,“linked”, “attached”, “complexed”, and “tethered,” and grammaticalequivalents, typically refer to two or more moieties connected with oneanother, either directly or indirectly (e.g., via one or more additionalmoieties that serve as a linking agent), to form a structure that issufficiently stable so that the moieties remain connected under theconditions in which the structure is used, e.g., physiologicalconditions. In some embodiments, the moieties are attached to oneanother by one or more covalent bonds. In some embodiments, the moietiesare attached to one another by a mechanism that involves specific (butnon-covalent) binding (e.g. streptavidin/avidin interactions,antibody/antigen interactions, etc.). Alternatively or additionally, asufficient number of weaker interactions (non-covalent) can providesufficient stability for moieties to remain connected. Exemplarynon-covalent interactions include, but are not limited to, affinityinteractions, metal coordination, physical adsorption, host-guestinteractions, hydrophobic interactions, pi stacking interactions,hydrogen bonding interactions, van der Waals interactions, magneticinteractions, electrostatic interactions, dipole-dipole interactions,etc.

“Biomolecules”: The term “biomolecules”, as used herein, refers tomolecules (e.g., proteins, amino acids, peptides, polynucleotides,nucleotides, carbohydrates, sugars, lipids, nucleoproteins,glycoproteins, lipoproteins, steroids, etc.) whether naturally-occurringor artificially created (e.g., by synthetic or recombinant methods) thatare commonly found in cells and tissues. Specific classes ofbiomolecules include, but are not limited to, enzymes, receptors,neurotransmitters, hormones, cytokines, cell response modifiers such asgrowth factors and chemotactic factors, antibodies, vaccines, haptens,toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, andRNA.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe materials that do not elicit a substantial detrimental responsein vivo. In some embodiments, a substance is considered to be“biocompatible” if its addition to cells in vitro or in vivo results inless than or equal to about 50%, about 45%, about 40%, about 35%, about30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less thanabout 5% cell death.

“Biodegradable”: As used herein, the term “biodegradable” refers tosubstances that are degraded under physiological conditions. In someembodiments, a biodegradable substance is a substance that is brokendown by cellular machinery. In some embodiments, a biodegradablesubstance is a substance that is broken down by chemical processes.

“Complement”: As used herein, the terms “complement,” “complementary”and “complementarity,” refer to the pairing of nucleotide sequencesaccording to Watson/Crick pairing rules. For example, a sequence5′-GCGGTCCCA-3′ has the complementary sequence of 5′-TGGGACCGC-3′. Acomplement sequence can also be a sequence of RNA complementary to theDNA sequence. Certain bases not commonly found in natural nucleic acidsmay be included in the complementary nucleic acids including, but notlimited to, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA).

Complementary need not be perfect; stable duplexes may containmismatched base pairs, degenerative, or unmatched bases. Those skilledin the art of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

“Contemporaneous” and “non-contemporaneous”: As used herein, the terms“contemporaneous,” “contemporaneously,” or grammatical equivalents, meanthat multiple events occur or happen at the same time without adetectable or identifiable sequential order. As used herein, the terms“non-contemporaneous,” “non-contemporaneously,” or grammaticalequivalents, mean that multiple events occur or happen in a detectableor identifiable sequential order.

“Crude”: As used herein, the term “crude,” when used in connection witha biological sample, refers to a sample which is in a substantiallyunrefined state. For example, a crude sample can be cell lysates orbiopsy tissue sample. A crude sample may exist in solution or as a drypreparation.

“Encoding region,” “coding region,” or “barcoded region”: As usedherein, the terms “encoding region,” “coding region,” “barcoded region”,or grammatical equivalents, refer to a region on an object or substrate(e.g., particle) that can be used to identify the object or substrate(e.g., particle). These terms may be used inter-changeably. Typically,an encoding region of an object bears graphical and/or optical featuresassociated with the identity of the object. Such graphical and/oroptical features are also referred to as signature features of theobject. In some embodiments, an encoding region of an object bearsspatially patterned features (e.g., stripes with various shapes and/ordimensions, or a series of holes with various sizes) that give rise tovariable fluorescent intensities (of one or multiple wavelengths). Insome embodiments, an encoding region of an object bears various typeand/or amount of fluorophores or other detectable moieties, in variousspatial patterns, that give rise to variable fluorescent signals (e.g.,different colors and/or intensities) in various patterns.

“Functionalization: As used herein, the term “functionalization” refersto any process of modifying a material by bringing physical, chemical orbiological characteristics different from the ones originally found onthe material. Typically, functionalization involves introducingfunctional groups to the material. As used herein, functional groups arespecific groups of atoms within molecules that are responsible for thecharacteristic chemical reactions of those molecules. As used herein,functional groups include both chemical (e.g., ester, carboxylate,alkyl) and biological groups (e.g., adapter, or linker sequences).

“Hybridize”: As used herein, the term “hybridize” or “hybridization”refers to a process where two complementary nucleic acid strands annealto each other under appropriately stringent conditions. Oligonucleotidesor probes suitable for hybridizations typically contain 10-100nucleotides in length (e.g., 18-50, 12-70, 10-30, 10-24, 18-36nucleotides in length). Nucleic acid hybridization techniques are wellknown in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor Press,Plainview, N.Y. Those skilled in the art understand how to estimate andadjust the stringency of hybridization conditions such that sequenceshaving at least a desired level of complementary will stably hybridize,while those having lower complementary will not. For examples ofhybridization conditions and parameters, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994,Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus,N.J.

“Hydrodynamic diameter”: The term “hydrodynamic diameter”, as usedherein, generally refers to the effective diameter of a hydratedmolecule (e.g., macromolecules, colloids, or particles) in solution,corresponding to the diameter of a sphere with equal mobility insolution. In some embodiments, a hydrodynamic diameter is used todescribe the measured size of particles in solution. In certainembodiments, hydrodynamic diameter may be determined by dynamic lightscattering size measurement. For example, Zetasizer Nano ZS instrument(Malvern) can be used to measure the hydrodynamic diameter of particlesas demonstrated in the Example Section below.

“Inert region”: As used herein, the terms “inert region,” “inert spacer”or grammatical equivalents, when used in connection with a region on anobject (e.g., particle), refer to a region that is not detectable abovea pre-determined triggering threshold by a flow-through scanning devicesuch as a flow cytometer. Typically, an inert region or spacer is anon-functionalized region. For example, an inert region is a region notloaded with probes or other detectable moieties.

“Interrogate”: As used herein, the terms “interrogate,” “interrogating,”“interrogation” or grammatical equivalents, refer to a process ofcharacterizing or examining to obtain data.

“Labeled”: The terms “labeled” and “labeled with a detectable agent ormoiety” are used herein interchangeably to specify that an entity (e.g.,a nucleic acid probe, antibody, etc.) can be visualized, for examplefollowing binding to another entity (e.g., a nucleic acid, polypeptide,etc.). The detectable agent or moiety may be selected such that itgenerates a signal which can be measured and whose intensity is relatedto (e.g., proportional to) the amount of bound entity. A wide variety ofsystems for labeling and/or detecting proteins and peptides are known inthe art. Labeled proteins and peptides can be prepared by incorporationof, or conjugation to, a label that is detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical,chemical or other means. A label or labeling moiety may be directlydetectable (i.e., it does not require any further reaction ormanipulation to be detectable, e.g., a fluorophore is directlydetectable) or it may be indirectly detectable (i.e., it is madedetectable through reaction or binding with another entity that isdetectable, e.g., a hapten is detectable by immunostaining afterreaction with an appropriate antibody comprising a reporter such as afluorophore). Suitable detectable agents include, but are not limitedto, radionucleotides, fluorophores, chemiluminescent agents,microparticles, enzymes, colorimetric labels, magnetic labels, haptens,molecular beacons, aptamer beacons, and the like.

“Monodisperse”: As used herein, the terms “monodisperse” or “monosized”refer to a collection of objects that have substantially the same sizeand shape when in the context of particles, and substantially the samemass in the context of polymers. Conversely, a collection of objectsthat have an inconsistent size, shape and mass distribution are calledpolydisperse. Monodisperse particles are typically synthesized throughthe use of template-based synthesis.

“Object” or “substrate”: As used herein, the terms “object” and“substrate” are used interchangeably and refer to any discrete mass. Anobject or substrate can be a particle, bead, planar surface, phage,macromolecules, cell, micro-organism, and the like.

“Particle”: The term “particle,” as used herein, refers to a discreteobject. Such object can be of any shape or size. Composition ofparticles may vary, depending on applications and methods of synthesis.Suitable materials include, but are not limited to, plastics, ceramics,glass, polystyrene, methylstyrene, acrylic polymers, metal, paramagneticmaterials, thoria sol, carbon graphited, titanium dioxide, latex orcross-linked dextrans such as Sepharose, cellulose, nylon, cross-linkedmicelles and teflon. In some embodiments, particles can be optically ormagnetically detectable. In some embodiments, particles containfluorescent or luminescent moieties, or other detectable moieties. Insome embodiments, particles having a diameter of less than 1000nanometers (nm) are also referred to as nanoparticles.

“Polynucleotide”, “nucleic acid”, or “oligonucleotide”: The terms“polynucleotide”, “nucleic acid”, or “oligonucleotide” refer to apolymer of nucleotides. The terms “polynucleotide”, “nucleic acid”, and“oligonucleotide”, may be used interchangeably. Typically, apolynucleotide comprises at least three nucleotides. DNAs and RNAs arepolynucleotides. The polymer may include natural nucleosides (i.e.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,biologically modified bases (e.g., methylated bases), intercalatedbases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose), or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

“Probe”: As used herein, the term “probe” refers to a fragment of DNA orRNA of variable length (e.g., 3-1000 bases long), which is used todetect the presence of target nucleotide sequences that arecomplementary to the sequence in the probe. Typically, the probehybridizes to single-stranded nucleic acid (DNA or RNA) whose basesequence allows probe-target base pairing due to complementarity betweenthe probe and target.

“Secondary Structure”: As used herein, the term “secondary structure”,when used in connection with a nucleic acid structure, refers to anystructure formed by basepairing interactions within a single molecule orset of interacting molecules. Exemplary secondary structures includestem-loop or double helix.

“Signal”: As used herein, the term “signal” refers to a detectableand/or measurable entity. In certain embodiments, the signal isdetectable by the human eye, e.g., visible. For example, the signalcould be or could relate to intensity and/or wavelength of color in thevisible spectrum. Non-limiting examples of such signals include coloredprecipitates and colored soluble products resulting from a chemicalreaction such as an enzymatic reaction. In certain embodiments, thesignal is detectable using an apparatus. In some embodiments, the signalis generated from a fluorophore that emits fluorescent light whenexcited, where the light is detectable with a fluorescence detector. Insome embodiments, the signal is or relates to light (e.g., visible lightand/or ultraviolet light) that is detectable by a spectrophotometer. Forexample, light generated by a chemiluminescent reaction could be used asa signal. In some embodiments, the signal is or relates to radiation,e.g., radiation emitted by radioisotopes, infrared radiation, etc. Incertain embodiments, the signal is a direct or indirect indicator of aproperty of a physical entity. For example, a signal could be used as anindicator of amount and/or concentration of a nucleic acid in abiological sample and/or in a reaction vessel.

“Specific”: As used herein, the term “specific,” when used in connectionwith an oligonucleotide primer, refers to an oligonucleotide or primer,under appropriate hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% sequence identity. In some embodiments,a specific oligonucleotide or primer contains at least 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, ormore bases of sequence identity with a portion of the nucleic acid to behybridized or amplified when the oligonucleotide and the nucleic acidare aligned.

“Stem-loop”: As used herein, the term “stem-loop”, when used inconnection with a nucleic acid structure, refers to a structure causedby an intramolecular base pairing typically occurring in single-strandedDNA or in RNA. The structure is also known as a hairpin or hairpin loop.Typically, it occurs when two regions of the same strand, usuallycomplementary in nucleotide sequence when read in opposite directions,base-pair to form a double helix that ends in an unpaired loop,resulting in lollipop-shaped structure.

“Substantially”: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

“Substantially complementary”: As used herein, the term “substantiallycomplementary” refers to two sequences that can hybridize understringent hybridization conditions. The skilled artisan will understandthat substantially complementary sequences need not hybridize alongtheir entire length. In some embodiments, “stringent hybridizationconditions” refer to hybridization conditions at least as stringent asthe following: hybridization in 50% formamide, 5×SSC, 50 mM NaH₂PO₄, pH6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart′ssolution at 42° C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.;and washing with 0.2×SSC, 0.1% SDS at 45° C. In some embodiments,stringent hybridization conditions should not allow for hybridization oftwo nucleic acids which differ over a stretch of 20 contiguousnucleotides by more than two bases.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides, among other things, methods andcompositions for detecting and quantifying target nucleic acids viapost-hybridization labeling. In some embodiments, the present inventionprovides a method for detecting the presence and/or abundance of targetnucleic acids in a sample by (a) contacting a plurality of nucleic acidprobes with a sample, each nucleic acid probe comprising a capturingsequence for binding a target nucleic acid and an adjacent adaptersequence for binding a universal adapter; (b) incubating the pluralityof probes and the sample, in the presence of one or more universaladapters, under conditions that permit binding of both an individualtarget nucleic acid and an individual universal adapter to a sameindividual nucleic acid probe; (c) carrying out a reaction that allowscoupling of the individual universal adapter to the individual targetnucleic acid when hybridized to the same individual nucleic acid probe;(d) detecting the presence of the one or more universal adaptersassociated with the plurality of nucleic acid probes, thereby detectingthe presence of the target nucleic acids in the sample. Typically,universal adapters are labeled with detectable moieties or otherlabeling groups to facilitate detection. In some embodiments, theplurality of nucleic acid probes suitable for the invention are attachedto a substrate or object (e.g., microarray, or particle).

In addition, it is contemplated that such ligation-based approach (orother coupling approach) may be used to encode or otherwisefunctionalize various objects or substrates (e.g., particles). Thus, insome embodiments, the present invention provides methods andcompositions for universal encoding. In particular embodiments, thepresent invention provides a method of encoding an object (e.g.,particle) by (a) providing an object or a substrate (e.g., particle)containing one or more encoding regions with each encoding regionbearing one or more single-stranded polynucleotide templates; (b)providing a blend of detectably labeled (e.g., labeled with fluorophoresor other detectable moieties) and unlabeled single-stranded encodingadapters, wherein each individual encoding adapter contains a sequencedesigned to specifically bind a polynucleotide template; (c) incubatingthe object with the encoding adapters under conditions that allowindividual encoding adapters to bind their corresponding polynucleotidetemplates; and (d) coupling the encoding adapters to their correspondingpolynucleotide templates, thereby encoding the object or substrate. Insome embodiments, by varying the amount of labeled adapter versusunlabeled adapter (with the same or similar sequence), it is possible tocontrol the amount of signal generated (e.g., fluorescence) in eachencoding region. Alternatively or additionally, objects or substrates(e.g., particles) embedded with nucleic acid anchors in a probe regioncan be used to attach desired probes to functionalize the probe regionof objects or substrates (e.g., particles). In this manner, encoding andprobe functionalization can be achieved in a single reaction.

Thus, inventive methods according to the present invention enable theproduction of several batches of objects (e.g., particles) with uniquecodes and probes from a single batch of objects (e.g., particles) with auniversal architecture. For highly multiplexed assays, this greatlyreduces production time and cost compared to independent synthesisparticle batches for each target. Importantly, particles generated usingthis method can also be used with post-hybridization labeling approachfor highly effective nucleic acid (e.g., microRNA) detection andquantification described herein.

Various aspects of the invention are described in further detail in thefollowing subsections. The use of subsections is not meant to limit theinvention. Each subsection may apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Nucleic Acid Probes for Post-Hybridization Labeling

Nucleic acid probes suitable for the present invention are designed togenerate a detectable signal indicating the presence and capture ofnucleic acid targets, e.g., miRNA targets. Thus, in some embodiments, anucleic acid probe suitable for the present invention includes acapturing sequence for binding a target nucleic acid of interest and anadjacent adapter sequence for binding a universal adapter. According tothe invention, the capturing sequence and the adapter sequence areconfigured such that binding of both the target nucleic acid and theuniversal adapter to the nucleic acid probe permits joining of theuniversal adapter to the target nucleic acid. In some embodiments, onceboth the target nucleic acid and the universal adapter bound to thenucleic acid probe, the 3′ end of the target would abut the 5′ end ofthe universal adapter. In some embodiments, once both the target nucleicacid and the universal adapter bound to the nucleic acid probe, the 5′end of the target would abut the 3′ end of the universal adapter. Insome embodiments, the universal adapter may be joined, linked, attachedor coupled to the targeted nucleic acid by enzymatic or chemicalcoupling. In some embodiments, a DNA or RNA ligase is used to link theuniversal adapter to the target nucleic acid. In some embodiments, a T4DNA ligase is used to link the universal adapter to the target nucleicacid. In some embodiments, a common, detectable universal adapter can beused to label multiple targets in a single reaction.

Capturing Sequence

In some embodiments, a suitable capturing sequence is specific to atarget nucleic acid (e.g., DNA, mRNA, or microRNA). The term “specific”when used in connection with a hybridization probe refers to a sequencethat can bind to its target under stringent conditions but not to otherregions.

For example, a suitable capturing sequence may contain a sequencesubstantially complementary to a target sequence on a target nucleicacid, such as a microRNA. Typically, a capturing sequence is based on atarget-specific nucleotide sequence. In some embodiments, a capturingsequence may contain a sequence substantially complementary to asequence specific to an microRNA of interest, e.g., microRNAs indicativeof certain cancer, diabetes, Alzheimer's or other diseases including butnot limited to, let-7a, miR-21, miR-29b-2, miR-181b-1, miR-143, miR-145,miR-146a, miR-210, miR-221, miR-222, miR-10b, miR-15a, miR-16, miR-17,miR-18a, miR-19a, miR20a, miR-1, miR-29, miR-181, miR372, miR-373,miR-155, miR-101, miR-195, miR-29, miR-17-3p, miR-92a, miR-25, miR-223,miR-486, miR-223, mir-375, miR-99b, miR- 127, miR- 126, miR-184.

In some embodiments, a suitable capturing sequence may be designed todistinguish different variable species of target nucleic acids. Thepresent invention is particularly useful to distinguish among multiplespecies of target nucleic acids with identical sequences at one end andvariable sequences at the other end. Thus, in some embodiments, acapturing sequence can be designed to be complementary to a desiredvariable end nucleotide sequence. Only the binding of a desired targetspecies will have a perfectly matching 3′ end that abut the 5′ end ofthe adapter sequence thereby permitting ligation of the adapter to thetarget. Therefore, the detection of the universal adapter associatedwith the probe indicates the presence of the target nucleic acid withthe desired end variability in the sample. In particular embodiments,the present invention is used to distinguish a precursor-microRNA from amature microRNA. Typically, a precursor-microRNA and mature microRNAhave identical 5′ region but distinct 3′ region due to the cleavage ofthe 3′ arm from the precursor form during the maturation process. Inorder to specifically detect a mature microRNA, a capturing sequence maybe designed to be substantially complementary to the sequence at the 3′end of the mature microRNA. Therefore, only the binding of a correctmature microRNA to the capturing sequence would result in the perfectlymatching 3′ end of the microRNA abutting the 5′ end of the adaptersequence permitting ligation of the adapter sequence to the targetsequence.

In some embodiments, a capturing sequence for nucleic acid targetscontains up to 50 nucleotides (e.g., up to 25, 20, 18, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides). In someembodiments, a capturing sequence is also chosen to ensure that themelting temperature Tm is between 20-50 C in ligation buffer.

Adapter Sequence

Generally, an adapter sequence can be any sequence and length.Typically, an adapter sequence and length are designed to such that (1)the melting temperature is between about 10-20 C in ligation buffer, (2)the sequence is not significantly self-complementary in order to avoidformation of hairpin, other secondary structure or homodimer, and/or (3)complete DNA probes (with adapter and miRNA sequence) does not formappreciable hairpins or other secondary structures. In some embodiments,a suitable adapter sequence contains up to 20 nucleotides (e.g., up to19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1nucleotides).

In some embodiments, a suitable nucleic acid probe contains a 3′ cap toprevent or mitigate incidental ligation. Exemplary suitable 3′ capsinclude, but are not limited to, inverted dT, or 3′ phosphates. In someembodiments, a suitable nucleic acid probe contains a chemical anchor atthe 5′ or 3′ end such that the probe can be attached to a substrate.Suitable exemplary chemical anchor groups include, but are not limitedto, carboxy groups, amine groups, thiol groups, biotin, and/or azidegroups. In some embodiments, a suitable probe may contain a particularnucleic acid sequence for association of the probe with a particularsubstrate or a specific location of on a substrate. Typically, suchparticular nucleic acid sequence is predetermined to be complementary toa capturing sequence embedded on a desired location of a substrate. Insome embodiments, the capture of the nucleic acid probe at a desiredlocation is associated with the identity of the probe. Therefore, suchparticular nucleic acid sequences are also referred to as nucleic acidbarcode.

Suitable probes typically are of a length that is large enough tohybridize specifically with its target but not so large as to impede thehybridization process. The size may be dependent on the desired meltingtemperature of the target-probe complex or required specificity oftarget discrimination. In some embodiments, suitable probes containsabout 10-70 nucleotides (e.g., 10-60, 10-50, 10-40, 10-30, 10-25, 10-20,15-70, 15-60, 15-50, 15-40, 15-30, 15-25, 20-70, 20-60, 20-50, 20-40,20-30 nucleotides). Various methods and softwares available in the artcan be used to design specific probes.

Nucleic acid probes according to the invention may include naturalnucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

Universal Adapter

According to the invention, a suitable universal adapter contains asequence complementary to the adapter sequence of a correspondingnucleic acid probe such that, once the universal adapter bound to thenucleic acid probe, the 5′ or 3′ end of the adapter abuts the 3′ or 5′end of a target nucleic acid, respectively. Suitable lengths andsequences of a universal adaptor can be selected using methods wellknown and documented in the art. For example a suitable adapter maycontain between 1 and 25 nucleotides in length (e.g., 1-20, 1-18, 1-16,1-14, 1-12, 1-10, 5-20, 5-15, or 5-10 nucleotides).

Adapters may be DNA, RNA, or any type of nucleic acid analog. Thenucleotides in adapters may be natural nucleosides (i.e., adenosine,thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g.,2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine,and 2-thiocytidine), chemically modified bases, biologically modifiedbases (e.g., methylated bases), intercalated bases, modified sugars(e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose),or modified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages).

In some embodiments, a universal adapter is biotinylated. In someembodiments, a biotinylated universal adapter may be detected by astreptavidin reporter conjugated to a detectable moiety including, butnot limited to, phycoerythrin, PE-Cy5, PE-Cy5.5, PE-Cy7, APC, PerCP,quantum dots, fluorophores or other detectable entities as describedherein (see the “Detectable entities” section below). In someembodiments, a biotinylated universal adapter may be detected by astreptavidin reporter conjugated to enzyme for enzymatic signalgeneration. In some embodiments, a suitable streptavidin reporter isconjugated to Alkaline Phosphatase, beta-Galactosidase, horse radishperoxidase, or other enzyme capable of turning over detectable products.In some embodiments, enzymatic signal generation permitschemiluminescence, fluorescence, or chromogenic detection (see theDetectable entities section). In some embodiments, a universal adaptercontains a nucleotide tail (also referred to as spacer or linker) toextend the biotin or enzyme group away from the polymer backbone of thegel matrix to avoid possible steric hindrance. A suitable nucleotidetail (spacer or linker) may contain various sequences. In someembodiments, a poly(A) or poly(T) tail is used. In some embodiments, asuitable nucleotide (such as a poly(A)) tail contains up to 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 bases.

In some embodiments, a universal adapter is directly labeled withfluorophores or other detectable entities (see the “Detectable moieties”section).

In some embodiments, multiple universal adapters may be used to labelmultiple distinct target nucleic acids in one reaction. Typically, insuch cases, each individual universal adapter is labeled withdistinctively detectable moieties or is detected by distinctbiotin-streptavidin reporter system.

Exemplary detectable entities suitable for the present invention aredescribed below.

Detectable Entities

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides; fluorescent dyes;chemiluminescent agents (such as, for example, acridinum esters,stabilized dioxetanes, and the like); bioluminescent agents; spectrallyresolvable inorganic fluorescent semiconductors nanocrystals (i.e.,quantum dots); microparticles; metal nanoparticles (e.g., gold, silver,copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes;colorimetric labels (such as, for example, dyes, colloidal gold, and thelike); biotin; dioxigenin; haptens; and proteins for which antisera ormonoclonal antibodies are available.

In some embodiments, the detectable moiety is biotin. Biotin can bebound to avidins (such as streptavidin), which are typically conjugated(directly or indirectly) to other moieties (e.g., fluorescent moieties)that are detectable themselves.

Below are described some non-limiting examples of other detectablemoieties.

Fluorescent Dyes

In certain embodiments, a detectable moiety is a fluorescent dye.Numerous known fluorescent dyes of a wide variety of chemical structuresand physical characteristics are suitable for use in the practice of thepresent invention. A fluorescent detectable moiety can be stimulated bya laser with the emitted light captured by a detector. The detector canbe a charge-coupled device (CCD) or a confocal microscope, which recordsits intensity.

Suitable fluorescent dyes include, but are not limited to, fluoresceinand fluorescein dyes (e.g., fluorescein isothiocyanine or FITC,naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein,6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryldyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes(e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.),Texas Red, Texas Red-X, SPECTRUM RED™, SPECTRUM GREEN, cyanine dyes(e.g., CY-3™, CY-5™, CY-3.5™, CY-5.5™, etc.), ALEXA FLUOR dyes (e.g.,ALEXA FLUOR™ 350, ALEXA FLUOR™ 488, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546,ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 633, ALEXA FLUOR™ 660,ALEXA FLUOR™ 680, etc.), BODIPY™ dyes (e.g., BODIPY™ FL, BODIPY™ R6G,BODIPY™ TMR, BODIPY™ TR, BODIPY™ 530/550, BODIPY™ 558/568, BODIPY™564/570, BODIPY™ 576/589, BODIPY™ 581/591, BODIPY™ 630/650, BODIPY™650/665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), and thelike. For more examples of suitable fluorescent dyes and methods forcoupling fluorescent dyes to other chemical entities such as proteinsand peptides, see, for example, “The Handbook of Fluorescent Probes andResearch Products”, 9th Ed., Molecular Probes, Inc., Eugene, Oreg.Favorable properties of fluorescent labeling agents include high molarabsorption coefficient, high fluorescence quantum yield, andphotostability. In some embodiments, labeling fluorophores exhibitabsorption and emission wavelengths in the visible (i.e., between 400and 750 nm) rather than in the ultraviolet range of the spectrum (i.e.,lower than 400 nm).

A detectable moiety may include more than one chemical entity such as influorescent resonance energy transfer (FRET). Resonance transfer resultsan overall enhancement of the emission intensity. For instance, see Juet. al. (1995) Proc. Nat'l Acad. Sci. (USA) 92: 4347, the entirecontents of which are herein incorporated by reference. To achieveresonance energy transfer, the first fluorescent molecule (the “donor”fluor) absorbs light and transfers it through the resonance of excitedelectrons to the second fluorescent molecule (the “acceptor” fluor). Inone approach, both the donor and acceptor dyes can be linked togetherand attached to the oligo primer. Methods to link donor and acceptordyes to a nucleic acid have been described previously, for example, inU.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which areherein incorporated by reference. Donor/acceptor pairs of dyes that canbe used include, for example, fluorescein/tetramethylrohdamine,IAEDANS/fluroescein, EDANS/DABCYL, fluorescein/fluorescein, BODIPYFL/BODIPY FL, and Fluorescein/ QSY 7 dye. See, e.g., U.S. Pat. No.5,945,526 to Lee et al. Many of these dyes also are commerciallyavailable, for instance, from Molecular Probes Inc. (Eugene, Oreg.).Suitable donor fluorophores include 6-carboxyfluorescein (FAM),tetrachloro-6-carboxyfluorescein (TET),2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and thelike.

A suitable detectable moiety can be an intercalating DNA/RNA dye thathave dramatic fluorescent enhancement upon binding to double-strandedDNA/RNA. Examples of suitable dyes include, but are not limited to,SYBR™ and Pico Green (from Molecular Probes, Inc. of Eugene, Oreg.),ethidium bromide, propidium iodide, chromomycin, acridine orange,Hoechst 33258, Toto-1, Yoyo-1, and DAPI (4′,6-diamidino-2-phenylindolehydrochloride). Additional discussion regarding the use of intercalationdyes is provided by Zhu et al., Anal. Chem. 66:1941-1948 (1994), whichis incorporated by reference in its entirety.

Enzymes

In certain embodiments, a detectable moiety is an enzyme. Examples ofsuitable enzymes include, but are not limited to, those used in anELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase,alkaline phosphatase, etc. Other examples include beta-glucuronidase,beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may beconjugated to a molecule using a linker group such as a carbodiimide, adiisocyanate, a glutaraldehyde, and the like.

Radioactive Isotopes

In certain embodiments, a detectable moiety is a radioactive isotope.For example, a molecule may be isotopically-labeled (i.e., may containone or more atoms that have been replaced by an atom having an atomicmass or mass number different from the atomic mass or mass numberusually found in nature) or an isotope may be attached to the molecule.Non-limiting examples of isotopes that can be incorporated intomolecules include isotopes of hydrogen, carbon, fluorine, phosphorous,copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium,bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F,32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, 111In, 125I, 123I, 129I, 131I,135I, 186Re, 187Re, 201Tl, 212Bi, 213Bi, 211At, 153Sm, 177Lu).

In some embodiments, signal amplification is achieved using labeleddendrimers as the detectable moiety (see, e.g., Physiol Genomics3:93-99, 2000), the entire contents of which are herein incorporated byreference in their entirety. Fluorescently labeled dendrimers areavailable from Genisphere (Montvale, N.J.). These may be chemicallyconjugated to the oligonucleotide primers by methods known in the art.

Substrates

In some embodiments, a nucleic acid probe suitable forpost-hybridization labeling is attached to a substrate or object.Suitable substrates or objects may have a planer, spherical ornon-spherical morphologies. Suitable substrates or objects may be solid,semi-solid, polymer, emulsion, or the like. Suitable substrates orobjects include, but are not limited to, microarrays, glasses, slides,particles, beads, films, membranes, microspheres (e.g., glass, polymer,etc.) with exterior or interior surface, cells including any geneticallyengineered cells, micro-organisms (e.g., C. elegans (e.g., engineerednematodes for drug testing), bacteria, yeast, and/or fungi) includingany genetically engineered micro-organisms.

For illustration purposes, particles are used in various embodimentsbelow.

Particles

Particles suitable for use in accordance with the present invention canbe made of any materials. Suitable particles can be biocompatible,non-biocompatible. Suitable particles can also be biodegradable ornon-biodegradable.

Materials

In some embodiments, particles are made of polymers. Exemplary polymersinclude, but are not limited to, poly(arylates), poly(anhydrides),poly(hydroxy acids), polyesters, poly(ortho esters), poly(alkyleneoxides), polycarbonates, poly(propylene fumerates), poly(caprolactones),polyamides, polyamino acids, polyacetals, polylactides, polyglycolides,poly(dioxanones), polyhydroxybutyrate, polyhydroxyvalyrate, poly(vinylpyrrolidone), polycyanoacrylates, polyurethanes and polysaccharides. Insome embodiments, polymers of particles include polyethylene glycol(PEG). In some embodiments, polymers of particles may be formed by stepor chain polymerization. The amount and kind of radical initiator, e.g.,photo-active initiator (e.g., UV or infrared), thermally-activeinitiator, or chemical initiator, or the amount of heat or lightemployed, may be used to control the rate of reaction or modify themolecular weight. Where desired, a catalyst may be used to increase therate of reaction or modify the molecular weight. For example, a strongacid may be used as a catalyst for step polymerization. Trifunctionaland other multifunctional monomers or cross-linking agents may also beused to increase the cross-link density. For chain polymerizations, theconcentration of a chemical initiator in a mixture of one or moremonomers may be adjusted to manipulate final molecular weight.

Exemplary methods for making particles are described in U.S. Pat. No.7,709,544 and US Application Publication No.: 20080176216, the entirecontents of which are incorporated herein by reference. For example,processes as discussed can be conducted with any polymerizableliquid-phase monomer in which shapes of particles suitable for use inthe present invention, can be defined and polymerized in a singlelithography-polymerization step. Exemplary monomers include AllylMethacrylate, Benzyl Methylacrylate, 1,3-Butanediol Dimethacrylate,1,4-Butanediol Dimethacrylate, Butyl Acrylate, n-Butyl Methacrylate,Diethyleneglycol Diacrylate, Diethyleneglycol Dimethacrylate, EthylAcrylate, Ethyleneglycol Dimethacrylate, Ethyl Methacrylate, 2-EthylHexyl Acrylate, 1,6-Hexanediol Dimethacrylate, 4-Hydroxybutyl Acrylate,Hydroxyethyl Acrylate, 2-Hydroxyethyl Methacrylate, 2-HydroxypropylAcrylate, Isobutyl Methacrylate, Lauryl Methacrylate, Methacrylic Acid,Methyl Acrylate, Methyl Methacrylate, Monoethylene Glycol,2,2,3,3,4,4,5,5-Octafluoropentyl Acrylate, Pentaerythritol Triacrylate,Polyethylene Glycol (200) Diacrylate, Polyethylene Glycol (400)Diacrylate, Polyethylene Glycol (600) Diacrylate, Polyethylene Glycol(200) Dimethacrylate, Polyethylene Glycol (400) Dimethacrylate,Polyethylene Glycol (600) Dimethacrylate, Stearyl Methacrylate,Triethylene Glycol, Triethylene Glycol Dimethacrylate,2,2,2-Trifluoroethyl 2-methylacrylate, Trimethylolpropane Triacrylate,Acrylamide, N,N,-methylene-bisacryl-amide, Phenyl acrylate, Divinylbenzene, etc. In certain embodiments, a monomer is characterized by apolymerization reaction that can be terminated with a terminationspecies. The terminating species, lithographic illumination, and monomerconstituents are therefore selected in cooperation to enable makingparticles suitable for use in the present invention.

In some embodiments, particles are hydrogels. In general, hydrogelscomprise a substantially dilute crosslinked network. Water or otherfluids can penetrate in the network forming such a hydrogel. In someembodiments, hydrogels suitable for use in the present invention aremade of or comprise a hydrophilic polymer. For example, hydrophilicpolymers may comprise anionic groups (e.g. phosphate group, sulphategroup, carboxylate group); cationic groups (e.g. quaternary aminegroup); or polar groups (e.g. hydroxyl group, thiol group, amine group).In some embodiments, hydrogels are superabsorbent (e.g. they can containover 99% water) and possess a degree of flexibility very similar tonatural tissue, due to their significant water content. Both of weightand volume, hydrogels are fluid in composition and thus exhibitdensities to those of their constituent liquids (e.g., water). Thepresent invention encompasses the recognition that hydrogels areparticularly useful in some embodiments of the present invention.Without wishing to be bound to any particular theory, it is contemplatedthat hydrogels enable 1) ease of implementation with detectioninstruments, in particular, commercially available instruments withoutsubstantial modifications (e.g., flow cytometers), and 2) ease ofincorporation of functional moieties (e.g., in a singlelithography-polymerization step) without requiring surfacefunctionalization. Due to their bio-friendly nature, hydrogels have beenused extensively in the fields of tissue engineering, drug delivery, andbiomolecule separation.

Various additional materials and methods can be used to synthesizeparticles. In some embodiments, particles may be made of or comprise oneor more polymers. Polymers used in particles may be natural polymers orunnatural (e.g. synthetic) polymers. In some embodiments, polymers canbe linear or branched polymers. In some embodiments, polymers can bedendrimers. Polymers may be homopolymers or copolymers comprising two ormore monomers. In terms of sequence, copolymers may be block copolymers,graft copolymers, random copolymers, blends, mixtures, and/or adducts ofany of the foregoing and other polymers.

In some embodiments, particles of the present invention may be made ofor comprise a natural polymer, such as a carbohydrate, protein, nucleicacid, lipid, etc. In some embodiments, natural polymers may besynthetically manufactured. Many natural polymers, such as collagen,hyaluronic acid (HA), and fibrin, which derived from various componentsof the mammalian extracellular matrix can be used in particles of thepresent invention. Collagen is one of the main proteins of the mammalianextracellular matrix, while HA is a polysaccharide that is found innearly all animal tissues. Alginate and agarose are polysaccharides thatare derived from marine algae sources. Some advantages of naturalpolymers include low toxicity and high biocompatibility.

In some embodiments, a polymer is a carbohydrate. In some embodiments, acarbohydrate may be a monosaccharide (i.e. simple sugar). In someembodiments, a carbohydrate may be a disaccharide, oligosaccharide,and/or polysaccharide comprising monosaccharides and/or theirderivatives connected by glycosidic bonds, as known in the art. Althoughcarbohydrates that are of use in the present invention are typicallynatural carbohydrates, they may be at least partially-synthetic. In someembodiments, a carbohydrate is a derivatized natural carbohydrate.

In certain embodiments, a carbohydrate is or comprises a monosaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is or comprises a disaccharide, including but not limitedto lactose, sucrose, maltose, trehalose, and cellobiose. In certainembodiments, a carbohydrate is or comprises a polysaccharide, includingbut not limited to hyaluronic acid (HA), alginate, heparin, agarose,chitosan, N,O-carboxylmethylchitosan, chitin, cellulose,microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),hydroxycellulose (HC), methylcellulose (MC), pullulan, dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, starch, heparin, konjac, glucommannan, pustulan, curdlan, andxanthan. In certain embodiments, the carbohydrate is a sugar alcohol,including but not limited to mannitol, sorbitol, xylitol, erythritol,maltitol, and lactitol.

In some embodiments, particles of the present invention may be made ofor comprise synthetic polymers, including, but not limited to,poly(arylates), poly(anhydrides), poly(hydroxy acids), poly(alkyleneoxides), poly(propylene fumerates), polymethacrylates polyacetals,polyethylenes, polycarbonates (e.g. poly(1,3-dioxan-2-one)),polyanhydrides (e.g. poly(sebacic anhydride)), polyhydroxyacids (e.g.poly((3-hydroxyalkanoate)), polypropylfumarates, polycaprolactones,polyamides (e.g. polycaprolactam), polyacetals, polyethers, polyesters(e.g. polylactide, polyglycolide, poly(dioxanones),polyhydroxybutyrate,), poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polyamines and copolymers thereof.Exemplary polymers also include polyvalerolactone, poly(sebacicanhydride), polyethylene glycol, polystyrenes, polyhydroxyvalyrate,poly(vinyl pyrrolidone) poly(hydroxyethyl methacrylate) (PHEMA),poly(vinyl alcohol) (PVA), and derivatives and copolymers thereof.

In some embodiments, polymers of particles may be formed by step orchain polymerization. The amount and kind of radical initiator, e.g.,photo-active initiator (e.g., UV or infrared), thermally-activeinitiator, or chemical initiator, or the amount of heat or lightemployed, may be used to control polymerization rate or modify molecularweights of resulting polymers. Where desired, a catalyst may be used toincrease the rate of reaction or modify the molecular weight. Forexample, a strong acid may be used as a catalyst for steppolymerization. Trifunctional and other multifunctional monomers orcross-linking agents may also be used to increase cross-link density ofpolymers. For chain polymerizations, the concentration of a chemicalinitiator in a mixture of one or more monomers may be adjusted tomanipulate final molecular weight.

In some embodiments, photocrosslinking methods are utilized to makepolymeric particles in accordance with the present invention.Photoinitiators produce reactive free radical species that initiate thecrosslinking and/or polymerization of monomers upon exposure to light.Any photoinitiator may be used in the crosslinking and/or polymeriationreaction. Photoinitiated polymerizations and photoinitiators arediscussed in detail in Rabek, Mechanisms of Photophysical Processes andPhotochemical Reactions in Polymers, New York: Wiley & Sons, 1987;Fouassier, Photoinitiation, Photopolymerization, and Photocuring,Cincinnati, Ohio: Hanser/Gardner; Fisher et al., 2001, Annu. Rev. Mater.Res., 31:171. A photoinitiator may be designed to produce free radicalsat any wavelength of light. In certain embodiments, the photoinitiatoris designed to work using UV light (200-500 nm). In certain embodiments,long UV rays are used. In other embodiments, short UV rays are used. Insome embodiments, a photoinitiator is designed to work using visiblelight (400-800 nm). In certain embodiments, a photoinitiator is designedto work using blue light (420-500 nm). In some embodiments, thephotinitiator is designed to work using IR light (800-2500 nm). Theoutput of light can be controlled to provide greater control over thecrosslinking and/or polymerization reaction. Control over polymerizationin turn results in control over characteristics and/or properties of theresulting hydrogel.

In some embodiments, particle can be or comprises inorganic polymer suchas silica (SiO₂). In some embodiments, particles according to theinvention are silica-based. For example, silicate materials may beuseful for the present applications due to their biocompatibility, easeof production and functionalization, and large surface-to-volume ratio.Silica-based particles such as porous silica particles, and any modifiedor hybrid particles can be of use in accordance with the presentinvention.

As well known in the art, silica-based particles may be made by avariety of methods. Some methods utilize the Stoller synthesis whichinvolves hydrolysis of tetraethoxyorthosilicate (TEOS) catalyzed byammonia in water/ethanol mixtures, or variations thereof. In someembodiments, silica-based particles are synthesized using known sol-gelchemistry, e.g., by hydrolysis of a silica precursor or precursors.Silica precursors can be provided as a solution of a silica precursorand/or a silica precursor derivative. Hydrolysis can be carried outunder alkaline (basic) or acidic conditions. For example, hydrolysis canbe carried out by addition of ammonium hydroxide to a solutioncomprising one or more silica precursor and/or derivatives. Silicaprecursors are compounds which under hydrolysis conditions can formsilica. Examples of silica precursors include, but are not limited to,organosilanes such as, for example, tetraethoxysilane (TEOS),tetramethoxysilane (TMOS) and the like. In some embodiments, silicaprecursor has a functional group. Examples of such silica precursorsincludes, but is not limited to, isocyanatopropyltriethoxysilane(ICPTS), aminopropyltrimethoxysilane (APTS),mercaptopropyltrimethoxysilane (MPTS), and the like. In someembodiments, microemulsion procedures can be used to synthesizeparticles suitable for use in the present invention. For example, awater-in-oil emulsion in which water droplets are dispersed as nanosizedliquid entities in a continuous domain of oil and surfactants and serveas nanoreactors for nanoparticle synthesis offer a convenient approach.

In some embodiments, particles may contain detectable moieties thatgenerate fluorescent, luminescent and/or scatter signal. In certainembodiments, particles contain quantum dots (QDs). QDs are bright,fluorescent nanocrystals with physical dimensions small enough such thatthe effect of quantum confinement gives rise to unique optical andelectronic properties. Semiconductor QDs are often composed of atomsfrom groups II-VI or III-V in the periodic table, but other compositionsare possible. By varying their size and composition, the emissionwavelength can be tuned (i.e., adjusted in a predictable andcontrollable manner) from the blue to the near infrared. QDs generallyhave a broad absorption spectrum and a narrow emission spectrum. Thusdifferent QDs having distinguishable optical properties (e.g., peakemission wavelength) can be excited using a single source. In general,QDs are brighter and photostable than most conventional fluorescentdyes. QDs and methods for their synthesis are well known in the art(see, e.g., U.S. Pat. Nos. 6,322,901; 6,576,291; and 6,815,064; all ofwhich are incorporated herein by reference). QDs can be rendered watersoluble by applying coating layers comprising a variety of differentmaterials (see, e.g., U.S. Pat. Nos. 6,423,551; 6,251,303; 6,319,426;6,426,513; 6,444,143; and 6,649,138; all of which are incorporatedherein by reference). For example, QDs can be solubilized usingamphiphilic polymers. Exemplary polymers that have been employed includeoctylamine-modified low molecular weight polyacrylic acid,polyethylene-glycol (PEG)-derivatized phospholipids, polyanhydrides,block copolymers, etc.

Exemplary QDs suitable for use in accordance with the present inventionin some embodiments, includes ones with a wide variety of absorption andemission spectra and they are commercially available, e.g., from QuantumDot Corp. (Hayward Calif.; now owned by Invitrogen) or from EvidentTechnologies (Troy, N.Y.). For example, QDs having peak emissionwavelengths of approximately 525 nm, approximately 535 nm, approximately545 nm, approximately 565 nm, approximately 585 nm, approximately 605nm, approximately 655 nm, approximately 705 nm, and approximately 800 nmare available. Thus QDs can have a range of different colors across thevisible portion of the spectrum and in some cases even beyond.

In certain embodiments, optically detectable particles are or comprisemetal particles. Metals of use include, but are not limited to, gold,silver, iron, cobalt, zinc, cadmium, nickel, gadolinium, chromium,copper, manganese, palladium, tin, and alloys thereof. Oxides of any ofthese metals can be used.

Certain metal particles, referred to as plasmon resonant particles,exhibit the well known phenomenon of plasmon resonance. The features ofthe spectrum of a plasmon resonant particle (e.g., peak wavelength)depend on a number of factors, including the particle's materialcomposition, the shape and size of the particle, the refractive index ordielectric properties of the surrounding medium, and the presence ofother particles in the vicinity. Selection of particular particleshapes, sizes, and compositions makes it possible to produce particleswith a wide range of distinguishable optically detectable propertiesthus allowing for concurrent detection of multiple analytes by usingparticles with different properties such as peak scattering wavelength.

Magnetic properties of particles can be used in accordance with thepresent invention. Particles in some embodiments are or comprisemagnetic particles, that is, magnetically responsive particles thatcontain one or more metals or oxides or hydroxides thereof. Magneticparticles may comprise one or more ferrimagnetic, ferromagnetic,paramagnetic, and/or superparamagnetic materials. Useful particles maybe made entirely or in part of one or more materials selected from thegroup consisting of: iron, cobalt, nickel, niobium, magnetic ironoxides, hydroxides such as maghemite (γ-Fe₂O₃), magnetite (Fe₃O₄),feroxyhyte (FeO(OH)), double oxides or hydroxides of two- orthree-valent iron with two- or three-valent other metal ions such asthose from the first row of transition metals such as Co(II), Mn(II),Cu(II), Ni(II), Cr(III), Gd(III), Dy(III), Sm(III), mixtures of theafore-mentioned oxides or hydroxides, and mixtures of any of theforegoing. See, e.g., U.S. Pat. No. 5,916,539 (incorporated herein byreference) for suitable synthesis methods for certain of theseparticles. Additional materials that may be used in magnetic particlesinclude yttrium, europium, and vanadium.

Size and Shape

In general, particles suitable for the present invention can be of anysize. In some embodiments, suitable particles have a greatest dimension(e.g. diameter) of less than 1000 micrometers (μm). In some embodiments,suitable particles have a greatest dimension of less than 500 μm. Insome embodiments, suitable particles have a greatest dimension of lessthan about 250 μm. In some embodiments, suitable particles have agreatest dimension (e.g. diameter) of less than about 200 μm, about 150μm, about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm,about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 10 μm. Insome embodiments, suitable particles have a greatest dimension of lessthan 1000 nm. In some embodiments, suitable particles have a greatestdimension of less than 500 nm. In some embodiments, suitable particleshave a greatest dimension of less than about 250 nm. In someembodiments, a greatest dimension is a hydrodynamic diameter.

Suitable particles can have a variety of different shapes including, butnot limited to, spheres, oblate spheroids, cylinders, ovals, ellipses,shells, cubes, cuboids, cones, pyramids, rods (e.g., cylinders orelongated structures having a square or rectangular cross-section),tetrapods (particles having four leg-like appendages), triangles,prisms, etc. In some embodiments, particles are rod-shaped. In someembodiments, particles are bar-shaped. In some embodiments, particlesare bead-shaped. In some embodiments, particles are column-shaped. Insome embodiments, particles are ribbon or chain-like. In someembodiments, particles can be of any geometry or symmetry. For example,planar, circular, rounded, tubular, ring-shaped, tetrahedral, hexagonal,octagonal particles, particles of other regular geometries, and/orparticles of irregular geometries can also be used in the presentinvention. Additional suitable particles with various sizes and shapesare disclosed in U.S. Pat. No. 7,709,544 and U.S. Pat. No. 7,947,487 andcan be used in the present invention, which are incorporated herein byreference.

Particles may have various aspect ratios of their dimensions, such aslength/width, length/thickness, etc. Particles, in some embodiments, canhave at least one dimension, such as length, that is longer than anotherdimension, such as width. According to the present invention, particleshaving at least one aspect ratio greater than one may be particularlyuseful in flow-through scanning (e.g., in a flow cytometer) tofacilitate their self-alignment. In some embodiments, particles may haveat least one aspect ratio of at least 1.5:1, at least 2:1, at least2.5:1, at least 3:1, at least 5:1, at least 10:1, at least 15:1, or evengreater.

It is often desirable to use a population of particles that isrelatively uniform in terms of size, shape, and/or composition so thateach particle has similar properties. In some embodiments, a populationof particles with homogeneity with diameters (e.g., hydrodynamicdiameters) are used. As used herein, a population of particles withhomogeneity with diameters (e.g., hydrodynamic diameters) refers to apopulation of particles with at least about 80%, at least about 90%, orat least about 95% of particles with a diameter (e.g., hydrodynamicdiameter) that falls within 5%, 10%, or 20% of the average diameter(e.g., hydrodynamic diameter). In some embodiments, the average diameter(e.g., hydrodynamic diameter) of a population of particles withhomogeneity with diameters (e.g., hydrodynamic diameters) ranges asdiscussed above. In some embodiments, a population of particles withhomogeneity with diameters (e.g., hydrodynamic diameters) refers to apopulation of particles that has a polydispersity index less than 0.2,0.1, 0.05, 0.01, or 0.005. For example, polydispersity index ofparticles used in accordance with the present invention is in a range ofabout 0.005 to about 0.1. Without wishing to be bound by any theory, itis contemplated that particles with homogeneity (e.g., with respect toparticle size) may have higher repeatability and can produce moreaccuracy in the present application. In some embodiments, a populationof particles may be heterogeneous with respect to size, shape, and/orcomposition.

Particles can be solid or hollow and can comprise one or more layers(e.g., nanoshells, nanorings, etc.). Particles may have a core/shellstructure, wherein the core(s) and shell(s) can be made of differentmaterials. Particles may comprise gradient or homogeneous alloys.Particles may be composite particles made of two or more materials, ofwhich one, more than one, or all of the materials possesses magneticproperties, electrically detectable properties, and/or opticallydetectable properties.

Particles may have a coating layer. Use of a biocompatible coating layercan be advantageous, e.g., if the particles contain materials that aretoxic to cells. Suitable coating materials include, but are not limitedto, natural proteins such as bovine serum albumin (BSA), biocompatiblehydrophilic polymers such as polyethylene glycol (PEG) or a PEGderivative, phospholipid-(PEG), silica, lipids, polymers, carbohydratessuch as dextran, other nanoparticles that can be associated withinventive nanoparticles etc. Coatings may be applied or assembled in avariety of ways such as by dipping, using a layer-by-layer technique, byself-assembly, conjugation, etc. Self-assembly refers to a process ofspontaneous assembly of a higher order structure that relies on thenatural attraction of the components of the higher order structure(e.g., molecules) for each other. It typically occurs through randommovements of the molecules and formation of bonds based on size, shape,composition, or chemical properties. In some embodiments, particles withcoating are also referred to as functionalized particles or surfacetreated particles.

In certain embodiments of the invention, a particle is porous, by whichis meant that the particle contains holes or channels, which aretypically small compared with the size of a particle. For example aparticle may be a porous silica particle, e.g., a porous silicananoparticle or may have a coating of porous silica. Particles may havepores ranging from about 1 nm to about 200 nm in diameter, e.g., betweenabout 1 nm and 50 nm in diameter. Between about 10% and 95% of thevolume of a particle may consist of voids within the pores or channels.

In some embodiments, particles may optionally comprise one or moredispersion media, surfactants, release-retarding ingredients, or otherpharmaceutically acceptable excipient. In some embodiments, particlesmay optionally comprise one or more plasticizers or additives.

In various embodiments, particles described herein may have at least oneregion bearing one or more probes described herein. In some embodiments,particles may have at least one encoded region. In some embodiments,particles have at least one encoded region and at least one regionbearing one or more probes. Such regions can be discrete regions ofsubstrates (objects) including particles used in accordance with thepresent invention. Each region, in some embodiments, can be optionallyfunctionalized. In various embodiments, particles described herein maybear an indicator for orientation (e.g., indicating coding region firstfollowed by probe region or vice versa).

Functionalization

Various methods known in the art (e.g., as discussed in U.S. Pat. No.7,709,544 and U.S. Pat. No. 7,947,487) and provided in the presentapplication are useful for functionalization of substrates or objects(e.g., particles) described herein.

Various functional moieties or groups may be introduced to the surfaceof the substrates that produce selected functionality (e.g., to captureencoding adapters, probes or target nucleic acids). Such functionalmoieties can be chemically attached to the surface, e.g., by covalentincorporation, or can be physically attached thereto or entrappedtherein.

In some embodiments, at least a portion of a substrate is made from amonomer. Such a monomer can be used alone or in combination withcopolymerized species to provide a selected functionality in theresulting substrate. For example, a functional moiety can be provided asa monomer or a part of a monomer that are polymerized, for example, by alithography-polymerization step of particle synthesis (see, U.S. Pat.No. 7,709,544 and U.S. Pat. No. 7,947,487 for details).

It is not intended that the present invention be limited to a particularcoding scheme. A signature for encoding can be a visually detectablefeature such as, for example, color, apparent size, or visibility (i.e.simply whether or not the particle is visible under particularconditions).

In many embodiments, graphical signatures and/or optically detectablesignatures are particularly useful in the present invention. In variousembodiments of the present invention, graphically encoding as discussedin U.S. Pat. No. 7,947,487 and encoding (e.g., universal encoding) asdisclosed herein are used.

In some embodiments, a graphical signature for encoding is or comprisesone or more spatially patterned features. In some embodiments, spatiallypatterned features include a plurality of open and closed codingelements. Coding elements can be arranged in a two-dimensional grid.Coding elements can also have non-uniform shapes or sizes. In certainembodiments, spatially patterned features further include an orientationindicator.

Additionally or alternatively, an optical signature can be used inaccordance with the present invention. In some embodiments, an opticalsignature for encoding is or comprises a feature of an absorption,emission, reflection, refraction, interference, diffraction, dispersion,scattering, or any combination thereof.

In some embodiments, an optical signature is intrinsic to functionalizedsubstrates in accordance with the present invention. In someembodiments, an optical signature is introduced to functionalizedsubstrates. Such introduction can be done before, with or aftercontacting with a sample, generating a signal from such contacting,and/or detecting such a signal.

To give but one example, a functionalized substrate may carry afunctional moiety that is not itself detectable, but upon furtherinteraction with and/or modification by other moieties can becomedetectable. In some embodiments, such a functional moiety can be afunctional group or moiety to facilitate association between a substrateand other entities.

Thus, additionally or alternatively, substrate surface is functionalizedto introduce chemical functional moieties that are designed tofacilitate association between a substrate and other entities (e.g.,probes, encoding agents, etc.). Suitable functional moieties can beintroduced to a surface of substrates by covalent attachment. In someembodiments, coupling agents can be used with various substrates forfunctionalization. Exemplary coupling agents may include bifunctional,tri-functional, and higher functional coupling agents, which are wellknown in the art, such as MeSiCl₃, dioctylphthalate, polyethylene-glycol(PEG), etc. In some embodiments, substrates are functionalized bycovalent attachment of streptavidin onto their surface via aheterobifunctional cross-linker with a polyethylene-glycol (PEG) spacerarm. A variety of functionalization methods are known in the art and canbe used to practice the present invention.

In some embodiments, a substrate surface is functionalized byintroducing capturing or anchor oligonucleotides to facilitate capturingand immobilization of individual nucleic acid molecules such assingle-stranded polynucleotide templates, encoding adapters or probes.In some embodiments, capturing or anchor oligonucleotides can containsequences complementary to a universal sequence present on nucleic acidtemplate molecules. Exemplary capturing or anchor oligonucleotides cancontain various numbers of nucleotides. For example, suitableoligonucleotides may contain 1-50 nucleotides (e.g., 3-40, 3-30, 3-20,30-15, 3-10, 6-40, 6-30, 6-20, 6-10, 8-30, 8-20, 8-15, 10-30, 10-20,10-15 nucleotides). In some embodiments, suitable oligonucleotides maycontain 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, or 50nucleotides. Various methods are known in the art for design andsynthesize suitable capturing or anchor oligonucleotides and suchmethods are well within skills of ordinary artisan.

In some embodiments, capturing or anchor oligonucleotides may beseparately synthesized and attached to a substrate surface for use, e.g.as disclosed by Lund et al. Nucleic Adds Research, 16: 10861-10880(1988); Albretsen et al, Anal. Biochem., 189: 40-50 (1990); Wolf et al,Nucleic Acids Research, 15: 2911-2926 (1987); or Ghosh et al, NucleicAcids Research, 15: 5353-5372 (1987).

In some embodiments, the attachment is covalent in nature. In furtherembodiments, the covalent binding of the capturing or anchoroligonucleotides and nucleic acid template(s) to the substrate isinduced by a crosslinking agent such as for example1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),succinic anhydride, phenyldiisothiocyanate or maleic anhydride, or ahetero-bifunctional crosslinker such as for examplem-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl[4-iodoacethyl]aminobenzoate (STAB), Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC),N-y-maleimidobutyryloxy-succinimide ester (GMBS),Succinimidyl-4-[p-maleimidophenyl]butyrate (SMPB) and the sulfo(water-soluble) corresponding compounds.

In some embodiments, functionalized substrates bearing chemical groupsor capturing or anchor oligonucleotides are used for universal encodingand/or probe region functionalization.

Universal Encoding

Universal encoding enables the production of functionalized substrateswith a universal architecture, which can be further encoded to generatesubgroups of substrates with distinct barcode giving rise to distinctidentity. For highly multiplexed assays, this greatly reduces productiontime and cost compared to independent synthesis of subpopulations ofsubstrates for each target.

In some embodiments, a functionalized substrate comprises one or moreuniversal encoding regions. Such encoding regions may be separated byinert or nonfunctionalized regions. Typically, each universal encodingregion bearing one or more templates for capturing encoding adapters bycovalent link via the functional groups or by hybridization and/orligation to a capturing or anchor oligonucleotides on the functionalizedsurface. In some embodiments, a template is or comprises asingle-stranded polynucleotide. For example, such a single-strandedpolynucleotide can include a predetermined nucleotide sequence thatspecifically bind a desired encoding adapter. In some embodiments, atemplate further include a stem-loop structure (i.e., a hairpinstructure). Predetermined nucleotide sequences, in certain embodiments,may be adjacent to stem-loop structures to facilitate ligation betweenthe template and the encoding adapter. In such embodiments, an encodingadapter that binds the template typically does not form a secondarystructure. In some embodiments, a single stranded template does notforms a hairpin structure, while an encoding adapter does.

In general, a predetermined nucleotide sequence with any basecombinations or lengths can be used in accordance with the presentinvention. In some embodiments, a predetermined nucleotide sequence hasa length of 1, 2, 3 bases or more. In some embodiments, a predeterminednucleotide sequence has a length of or more than 4 bases, 5 bases, 6bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 base, 12 bases, 13 bases,14 bases, 15 bases, 20 bases, 25 bases or 30 bases. In some embodiments,a predetermined nucleotide sequence has a length in a range of any twovalues above. The length of predetermined nucleotide sequences can bethe same for one substrate or can vary from each other.

In some embodiments, single-stranded polynucleotide templates can beused to capture encoding adapters. Suitable encoding adapters may beDNA, RNA, or any type of nucleic acid analog. In many embodiments, anencoding adapter is or comprises a single-stranded polynucleotide. Insome embodiments, an encoding adapter comprises a nucleotide sequencethat is complementary to the predetermined sequence of a correspondingtemplate. Typically, an encoding adapter contains up to 30, 25, 20, 18,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides.

In some embodiments, encoding adapters, once bound to the template, canbe joined to the template by T4 DNA ligase or via other enzymatic orchemical coupling.

Encoding adapters can be labeled or unlabeled. In some embodiments,encoding adapters is labeled with a detectable moiety (e.g., anoptically detectable moiety). Various detectable moieties may be usedincluding fluorophores, chromophores, radioisotopes, quantum dots,nanoparticles and/or intercalating DNA/RNA dyes. Additional examples ofdetectable moieties are described in the Detectable Moieties sectionabove.

In various embodiments, encoding adapters used in accordance with thepresent invention is a blend of labeled and unlabeled encoding adapters.In some embodiments, the labeled and unlabeled encoding adapters havethe same or similar sequences and bind the same templates. In someembodiments, by varying the amount of labeled encoding adapters versusunlabeled encoding adapter, it is possible to control the amount ofsignal generated (e.g. fluorescence) in a region to achieve desiredlevel. In some embodiments, a lock sequence can be used to selectivelydictate which adapters will bind and be ligated to each hairpin proberegion. In this way, several stripes of independently addressablehairpin probe regions can be used for encoding.

In some embodiments, a signal of at least one labeled encoding adapteris used to determine the orientation of the substrate. In someembodiments, a signal of at least one labeled encoding adapter is usedto normalized detectable signals form other labeled encoding adapters.

It is possible to use multiple colors (or emission wavelengths ingeneral) when implementing the universal encoding scheme describedherein. This may be accomplished by using blends of universal adaptersmodified with varying species, such as fluorophores, with uniqueemission spectra. Depending on the amount of each adapter added to theligation mix, varying amounts will be ligated to the templates embeddedin the particles, allowing levels of multiple “colors” to be adjusted ineach encoding region. In one example, two fluorophores can be used togenerate two-color codes on particles/substrates as shown below, butmore colors can easily be used.

In some embodiments, fluorescence in each coding region can bedistinguishable at multiple levels, e.g., up to 10-20 levels (e.g., upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 levels). For example, when three encoding regions are used and 10levels are distinguishable for each, it would allow up to 1000(10×10×10) unique codes. Additionally or alternatively, multiple signals(e.g., different fluorescent colors) can be used for encoding. In someembodiments, each encoding region has one signal distinct from eachother. In some embodiments, substrates and encoding adapters can bedesigned such that at least one encoding region of the substrates isattached with one or more kinds of encoding adapters generating multiplesignals. In some embodiments, each encoding region has multiple signalsand by varying the amount of encoding adapters, a desired signal ratiocan be achieved for encoding.

Probe Region Functionalization

A substrate used in accordance with the present invention can compriseone or more probe-bearing regions in addition to encoding regions. Twotypical schematics for universal encoding and probe functionalizationare represented in FIG. 1 and FIG. 2.

In some embodiments, each probe region bears anchors for attachingprobes of interest via, e.g., ligation-based approach. Ligation can beperformed with three species (anchor, linker, and probe) or two species(hairpin anchor and probe). A schematic of probe-regionfunctionalization using three-species ligation, two-species ligation,and chemical modification is depicted in FIG. 3.

In some embodiments, probe region functionalization includes chemicalmodification, such as the use of peptide chemistry to attach aminatedprobes to carboxylated substrates using carbodiimide chemistry. Detailedexemplary methods for functionalization are shown in the Examplessection below.

Desired probes specific for target nucleic acids may be designed usingvarious methods known in the art. In some embodiments, desired probesfor probe region functionalization include nucleic acid probes forpost-hybridization labeling described herein.

In some embodiments, probe regions and encoding regions are separatedfrom one another by inert regions. In some embodiments, one or moreprobe-bearing regions and one or more encoding regions overlap with eachother. In some embodiments, an encoding and probe-bearing region can bethe same region.

In some embodiments, different detectable signals (e.g., differentfluorescent colors) may be used for encoding regions and probe-bearingregions. In some embodiments, same type of detectable signals are used,in particular, when encoding regions and probe-bearing regions areseparated from each other.

For two-species functionalization, it is possible to use linear anchorsand adapters that have hairpins. The adapter and anchor species may bedesigned to have minimal hairpin formation in ligation conditions orvary tightly bound hairpins. Detectable moieties for encoding mayinclude fluorophores, chromophores, radioactive species, magneticspecies, quantum dots, conductive materials, etc. Any number of codingregions may be used, and they need not be stripes. Any number of colorsor otherwise distinguishable signals may be included in each encodingregion. This approach may be used with other substrates including beads,planar surfaces, gel pads, etc. The substrates may be solid, polymer,emulsions, etc.

In addition to ligation based approach, inventive methods for universalencoding and/or functionalization can be implemented with other enzymesincluding ligases, polymerases, among others. For example, although T4DNA ligase was used in the experiments described below, it is possibleto use other enzymes to join oligonucleotides together. Other possibleenzymes include, but are not limited to, other DNA ligases, RNA ligases,polymerases, etc. In a slightly different approach, polymerases can alsobe used to extend oligonucleotides, using a desired nucleic acidtemplate, as means of adding nucleic acid probes for functionalizationor labeled species for encoding or detection (FIG. 4). Using thisapproach or ligation-based approaches, multiple probe regions can beadded to a single particle when multiple probe “anchors” are used.

Target Nucleic Acids

Methods and compositions described herein may be used to detect anytarget nucleic acids. In general, target nucleic acids may be any formof DNA, RNA, or any combination thereof. In certain embodiments of thepresent invention, a target nucleic acid may be or contain a portion ofa gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA,rRNA, microRNA, or any combination thereof.

A target nucleic acid, in various embodiments, can be one that is foundin a biological organism including, for example, a microorganism orinfectious agent, or any naturally occurring, bioengineered orsynthesized component thereof.

According to the present invention, provided compositions andmethodologies are particularly useful in quantifying transcript (e.g.,primary transcripts, mRNA, etc.) nucleic acids. In some embodiments,provided methods herein are used to detect and/or quantify miRNAs.miRNAs can be found in genomes of humans, animals, plants and viruses.According to the present invention, a target nucleic acid, in someembodiments, can be or comprise one or more miRNAs that is/are generatedfrom endogenous hairpin-shaped transcripts. In some embodiments, atarget nucleic acid can be or comprise one or more miRNAs that is/aretranscribed as long primary transcripts (pri-microRNAs), for example, byRNA polymerase II enzyme in animals. There are total 1424 human miRNAgenes currently listed in the miRNA database(http://microrna.sanger.ac.uk/sequences/ftp.shtml), which is equivalentto almost 3% of protein-coding genes. Many miRNAs are thought to beimportant in the regulation of gene expression. Typically, microRNAs areproduced in precursor form and then processed to mature form bytypically cleaving the 3′ arm of the precursor stem-loop structure.Therefore, a precursor microRNA and a mature microRNA have identical 5′end but distinct 3′ end. Selective end-labeling can be used to detectmature microRNA species without detection of precursor species bydesigning a capturing sequence complementary to the 3′ end sequence. Anexample of selective end-labeling is described in the examples section.

Any of a variety of biological samples may be suitable for use withmethods disclosed herein. Generally, any biological samples containingnucleic acids (e.g., cells, tissue, etc.) may be used. Types ofbiological samples include, but are not limited to, cells, tissue, wholeblood, plasma, serum, urine, stool, saliva, cord blood, chorionic villussamples amniotic fluid, and transcervical lavage fluid. Tissue biopsiesof any type may also be used. Cell cultures of any of theafore-mentioned biological samples may also be used in accordance withinventive methods, for example, chorionic villus cultures, amnioticfluid and/or amniocyte cultures, blood cell cultures (e.g., lymphocytecultures), etc. In some embodiments, biological specimens comprisediseased cells such cancer or tumor cells.

Thus, a typical biological sample suitable for the present inventioncontain heterogeneous nucleic acids. In some embodiments, a biologicalsample contains a mixture of nucleic acids from different cell types(e.g., normal cells and diseased cells such as tumor cells). In someembodiments, a biological sample (e.g., blood, serum or plasma) containsa mixture of maternal nucleic acids and fetal nucleic acids.

In some embodiments, the present invention is used to detect targetnucleic acids that are present in low abundance or as rare events in abiological sample. In some embodiments, target nucleic acids that may bedetected by an inventive method of the present invention are present ata concentration ranging from 0.1 amol-10,000 amol. In some embodiments,the target nucleic acids are present at a concentration below 10,000amol, below 5,000 amol, below 1,000 amol, below 800 amol, below 600amol, below 400 amol, below 200 amol, below 100 amol, below 50 amol,below 40 amol, below 30 amol, below 20 amol, below 10 amol, or below 1amol. In some embodiments, the amount of target nucleic acids detectedby an inventive method of the present invention represents less than 1%(e.g., less than 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%) of the totalnucleic acids in a biological sample. In some embodiments, the amount oftarget nucleic acids detected by an inventive method of the presentinvention represents less than 1% (e.g., less than 0.5%, 0.1%, 0.01%,0.001%, 0.0001%) of the total nucleic acids in a biological sample. Insome embodiments, the amount of target nucleic acids detected by aninventive method of the present invention represents less than 1 out ofa million of the total nucleic acids in a biological sample. In someembodiments, the amount of target nucleic acids detected by an inventivemethod of the present invention represents less than 1 out of 10 millionof the total nucleic acids in a biological sample. The target nucleicacids may be detected in crude sample or may be detected as isolated orpurified sample.

Scanning and Quantification

Substrates or objects described herein may be characterized usingvarious methods. In particular, various methods involving flow-throughscanning and/or static imaging can be used to detect substrates boundwith target nucleic acids and/or to determine amount of the targetnucleic acids. Typically, target nucleic acids attached to substratesare determined based on detection of signals. According to the presentinvention, signals “indicative of” a target nucleic acid are typicallyassociated with the identity of substrates or locations on substrates towhich the target nucleic acid is attached. For example, signals emanatefrom one or more detectably labeled probes or targets that becomesassociated with signals indicative of one or more encoding regions ofthe substrates bearing the probes or targets.

In some embodiments, signals indicative of target nucleic acids aregenerally distinguishable from signals indicative of identity ofsubstrates. In some embodiments, probes or universal adapters specificfor a target nucleic acid and encoding adapters for coding regions arelabeled with distinctively detectable signals. For example, probes oruniversal adapters specific for the target nucleic acid may be labeledwith fluorescent moieties that have a different emission spectrum (i.e.,color and wavelength) than that of the fluorescent moieties with whichthe coding regions are labeled. Thus, in some embodiments, substrates(e.g., particles) of the present invention can be scanned using amulti-scanning system involving more than one excitation sources anddetectors (see FIG. 5).

In some embodiments, single-color scanning is used. Signals indicativeof separate “code” and “probe” regions are used to identify substrates(e.g., particles) and capture targets, respectively. Using particles asexamples, as described in detail below, signal patterns from the coderegions (e.g., bearing holes, stripes, encoding adapters and/orcombination thereof) of a particle serve as the basis for a graphicalmultiplexing barcode to identify the probe(s) in a particular particle.In some embodiments, unlike traditional bead-based systems that useoptical encoding of spheres, an arrangement in which particles havemultiple distinct regions makes single-color scanning possible, withonly one excitation source and one detector required. In someembodiments, particles can bear graphical features (e.g., stripes,holes, or the like) with variable fluorescent intensities (of one ormultiple wavelengths), optical properties, dimensions, etc. (see FIG.6).

Particles are used as examples to illustrate the scanning andquantification process in more detail below. However, methods describedherein may be used with various other types of substrates or objects.

Interrogating Particles

In some embodiments, the present invention provides a method forcharacterizing multifunctional objects (e.g., particles) including oneor more steps of (a) interrogating a plurality of objects (e.g.,particles), wherein each individual object (e.g., particle) containingone or more interrogation regions detectable as a sequence of events;(b) recording multiple events, wherein each individual event correspondsto each individual interrogation region detectable above apre-determined triggering threshold; (c) grouping the recorded multipleevents, and (d) characterizing the plurality of objects based on thegrouped events.

In some embodiments, particles are interrogated using image analysis ineither static or flow-through settings. For high-throughputapplications, it is desirable to scan the particles rapidly, preferablyusing existing commercial equipment. For example, flow cytometers areparticularly useful for flow-through analysis of fluorescently labeledbeads and particles, providing means for particle alignment, preciseillumination, and accurate quantification of fluorescence emission. Insome embodiments, encoded multifunctional particles are designed suchthat they can be scanned using commercially-available or custom designedflow-through device, such as, flow cytometers.

In some embodiments, particles suitable for flow-through scanning areengineered to mimic a series of cells (e.g., 2, 3, 4, 5, or more) thatflow past an interrogation zone. In particular embodiments, outerregions (e.g., both end regions) of suitable particles are codingregions while one or more inner regions contain probes where the targetis captured. Each coding region and probe region can be interrogatedseparately (e.g., sequentially or non-contemporaneously) and each regionis also referred to as an interrogation region. In particularembodiments, rod-shaped particles that bear multiple interrogationregions are recorded as “events” using standard cytometery signalprocessing. By analyzing the sequence and time-proximity of such events,one can infer which ones belong in the same particle. These events canthen be analyzed to decode the particle and quantify target bound to theprobe region. Signal quantification can be achieved using fluorescence,light scattering, luminescence, etc.

Typically, raw signal is obtained from the cytometer detectors (orsignal processing boards) using standard cytometery software. The signalcan then be processed using custom software to import standard flowcytometery (FCS) files and reconstruct the events into particles andcorresponding probe and coding regions.

Various flow-cytometery and other flow-through reading devices may beused in accordance with the present invention, including variouscommercially available flow-cytometers and customly designed devices.Exemplary suitable flow cytometers include, but are not limited to,Millipore Guava 8HT, Guava 5HT, Accuri C6, BD FACSCalibur, and amongother cytometers.

Multiple-Event Particles

As a non-limiting example, when a particle travels through a cytometer'sflow cell, it is excited with an illumination spot while detectors areused to monitor several parameters including forward scatter and sidescatter of the illumination, and various wavelengths of emitted light.By setting a threshold on one of these parameters in a triggeringchannel, a user can define the instances that the cytometer softwarewill record as events. If the signal from the detector in the triggeringchannel increases beyond the threshold level set by the user, thecytometery hardware and software will start to record an event—measuringthe maximum signal height and integrated area from each detector whilethe triggering signal remains above the threshold. Events are typicallyreported with the height and area observed in each channel, along withthe event width and a time-stamp of when the event occurred.

Typically, a single particle or bead is recorded as a single event.However, in many embodiments, particles according to the invention(e.g., rod-shaped particles) with multiple functional regions can beread as a sequence of distinct events. This is accomplished by usingparticles that have functional regions (for example: fluorescent)separated by inert regions (for example: non fluorescent). Byincorporating threshold-triggering entities in the functional regions ofthe particles, but not in the inert regions, typical cytometery signalprocessing software records the functional regions as discrete events.This can be accomplished using entities that cause scatter orfluorescence. Such entities could include microparticles, nanoparticles,reflective monomers, metallic materials, fluorescently-labeled monomers,quantum dots, fluorescent dyes, carbon nanotubes, liquid crystals, andvarious detectable entities described herein.

An example is provided in the Examples section to illustrate how thisapproach works and the distinction from standard cytometery (Example 9).A example of particle scanning using a particular flow cytometery isprovided in Example 10.

Data Analysis

For data analysis, an algorithm can be written to group events intoparticles, orients the particles, normalizes fluorescence against astandard if desired, and quantifies the fluorescence, scatter, or eventwidth in each code and probe region. The corresponding code for eachparticle can then be given a confidence level, and those that were notcalled with a pre-defined level of confidence can be excluded from theanalysis. The fluorescence in the probe region can then be used todetermine the amount of target present in the sample analyzed. Thissystem can be easily automated using software that performed analysisduring or after scanning.

Grouping of Events

In some embodiments, events are grouped based on spatial andtemporal-proximity. In some embodiments, events are grouped based onpatterns of measured properties for each event.

Typically, each event recorded by the cytometer is given a timestampwith a pre-determined resolution of, e.g., 1 ms, based on the flow ratein each cytometery. For example, as particles typically move at rates of˜1 m/s through the flow cell, the interrogation of a particle that is200 μm long is expected to last ˜0.2 ms. As such, it can be expectedthat the two events recorded from a single multifunctional particlewould appear in the same timestamp.

In some embodiments, calibration beads are scanned fairly randomlythroughout the course of data acquisition. Typically, at least one eventis recorded for calibration beads. The multifunctional particles, on theother hand, typically show clustering of 2 or 4 events per timestamp,which lends very well to the theory that each particle is being read astwo events. In addition, it can be clearly seen from the plots of eventvs. time that during each timestamp, there is a high- and low-levelfluorescence reading. The particles were designed to have one bright andone dim region of fluorescence in the FL-2 channel, which also givessupport to the theory that each particle is being read as two discreteevents. This approach can be applied to three or more events perparticle as well. Each region/event can vary in terms of fluorescencelevel, forward or side scatter, and width.

It is possible to incorporate distinct levels of multiple fluorophoresinto each code region of the multifunctional particles. As aproof-of-concept, we used rod-shaped particles, 200×35×30 μm, with asingle 60 μm code region on one end. The code region was labeled usingfour distinct levels of Cy3 and Cy5 fluorescent dyes. Particles wereanalyzed using the Accuri C6 cytometer with a flow rate of 100 μl/min, acore size of 40 μm, and a threshold of 5000 on FL4. The results areshown in FIG. 7.

The plot in FIG. 7 shows that it is possible to create a distinctfluorescent fingerprint in each code region of multifunctionalparticles. Each cluster of data points represents a distinct code.

Reading of Raw Signal

In some embodiments, interrogating multifunctional particles in standardflow cytometers is to acquire signal from the cytometer detectors beforeit is processed into events by the machine's firmware and use customsoftware to identify, orient, and analyze particles scans.

In some embodiments, raw data files (e.g., 20 million points/scan)produced by the scanning process are analyzed with a custom writtenMATLAB algorithm designed to isolate individual particle signatures,identify the code displayed by each particle, and quantify the amount oftarget bound. The algorithm processed scans of 50 μl samples in under 5s, making the approach suitable for high-throughput applications. In theinitial filter step, the algorithm excised portions of the scan thatexceeded a threshold voltage and then interrogated each removed segmentfor characteristics that identified it as a particle signature. Usingspecific properties of the fluorescent code region as reference points,a high-confidence estimate of the velocity of each particle wasdetermined and utilized to pinpoint trough locations for the five codingholes. The orientation of the particle (i.e., probe- or code-first) wasestablished using the fixed-value “3” hole that bordered the inertbuffer region. After an initial code identity was calculated from thetrough depths, a secondary review was conducted by measuring thestandard deviation in trough depths of holes designated to be of thesame level and corrective action was taken if necessary. In the finaldecoding step, a confidence score was calculated for the particle bycomputing the linearity of the correlation between trough depth andassigned level. A particle decoding event was rejected if its Pearsoncoefficient fell below 0.97.

In order to calculate the amount of target bound, the measured particlevelocity was used to infer the location of the center of the proberegion. Briefly, a search window was used to investigate the scan inthis region, seeking to identify a local maximum that could becorrelated to a target-binding event. If a maximum was found, theposition of the search window along the scan profile was adjusted untilthe two endpoints were sufficiently close in signal amplitude, therebyselecting a nearly symmetrical portion of the maximum over which toaverage for quantification purposes. In the cases in which a maximum wasnot found, the original estimate of probe center was used to calculate amean signal without a search window. To calculate the background for agiven probe sequence and incubation condition, particles from the samesynthesis batch were incubated in the presence of, e.g., only 100 amolof miSpike target according to the procedure described above. Thismethod provided a measure of the probe-dependent background that arosefrom the PEG scaffold and the universal adapter used in the labelingprocess. Also, upon calculation of all code identities and targetlevels, a particle would be rejected from consideration if its targetlevel was more than one inter-quartile range above the third quartile orbelow the first quartile of the data set consisting of target levelsassociated with the probe in question.

Various examples of particle scanning and quantification are provided inthe Examples section. Additional scanning and quantification methods aredescribed in International Application entitled “ScanningMultifunctional Particles,” filed on even date, the disclosure of whichis incorporated herewith in its entirety.

Other Embodiments

There are several variations and alternate approaches to the embodimentsdescribed above. Although rod-shaped particles are used as examplesdescribed here, the present invention may be used to scan objects orparticles with many other morphologies as well. For instance, particlesmay be anisotropic, have a head on one side, include rounded shapes,have holes in them, etc. In some embodiments, the present invention maybe used to scan a variety of multifunctional entities including longnucleic acids, DNA origami, self-assembled structures, biologicalorganisms, string-like objects, ribbon-like objects, etc. Furthermore,any combination of information recorded by the cytometer for each event,including height, area, width, or any combination thereof can be usedfor encoding or target quantification.

Other commercially-available instruments are capable of readingparticles with multiple functional regions and can be used to practicethe present invention. One example is an instrument capable of measuringchanges in electrical conductance, or electrical resistance of a fluidicchannel such as a Coulter Counter. The resulting current or voltagegenerated by a particle by a detector in such systems can be used tocharacterize particle size, shape, chemical composition, or surfaceproperties. Additionally, laser-scanning cytometry (LSC), which allowshigh resolution visualization of particles in flow, may be used toidentify the identifier regions and probe regions on particles withseveral functionalized regions. Such LSC systems are commerciallyavailable from companies such as CompuCyte. There also exist commercialcytometers that image cells/particles as they pass (e.g. AmnisImageStream). These can be used with suitable image-processing softwareto decode particles and quantify target. In addition, it may be possibleto use non-fluorescent means of quantification such as surface-plasmonresonance or radiation.

Applications

The present invention has many applications, including, but not limitedto, diagnosis and prognosis of diseases, disorders or conditions basedon detection or quantification of a target nucleic acid (e.g., microRNA,DNA or mRNA) in a biological sample.

Those of ordinary skill reading the present disclosure, will appreciateits broad applicability. For example, the present invention can be usedto diagnose or prognose a variety of diseases including, but not limitedto, cancer (e.g., lung cancer, breast cancer, stomach cancer, pancreaticcancer, lymphoma, leukemia, colon cancer, liver cancer, etc.), diabetes,neurodegenerative diseases (e.g., Alzheimer's), infectious diseases,genetic diseases.

Representative bacterial infectious agents which can be detected and/ordetermined by the present invention include, but are not limited to,Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas,Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacteriumaviumintracellulare, Yersinia, Francisella, Pasteurella, Brucella,Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus,Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella,Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseriameningitides, Hemophilus influenza, Enterococcus faecalis, Proteusvulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium,Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens,Nocardia, and Acitnomycetes.

Representative fungal infectious agents which can be detected and/ordetermined by the present invention include, but are not limited to,Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasmacapsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candidaalbicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrixschenckii, Chromomycosis, and Maduromycosis.

Representative viral infectious agents which can be detected and/ordetermined by the present invention include, but are not limited to,human immunodeficiency virus, human T-cell lymphocytotrophic virus,hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis C Virus),Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxoviruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses,polio viruses, toga viruses, bunya viruses, arena viruses, rubellaviruses, and reo viruses.

Representative parasitic agents which can be detected and/or determinedby the present invention include, but are not limited to, Plasmodiumfalciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale,Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp.,Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp.,Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobiusvermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculusmedinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystiscarinii, and Necator americanis.

The present invention can also be useful for detection and/ordetermination of drug resistance by infectious agents. For example,vancomycin-resistant Enterococcus faecium, methicillin-resistantStaphylococcus aureus, penicillin-resistant Streptococcus pneumoniae,multi-drug resistant Mycobacterium tuberculosis, and AZT-resistant humanimmunodeficiency virus can be identified with the present invention.

Genetic diseases can also be detected and/or determined by the processof the present invention. This can be carried out by prenatal orpost-natal screening for chromosomal and genetic aberrations or forgenetic diseases. Examples of detectable genetic diseases include, butare not limited to: 21 hydroxylase deficiency, cystic fibrosis, FragileX Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndromeor other trisomies, heart disease, single gene diseases, HLA typing,phenylketonuria, sickle cell anemia, Tay-Sachs Disease, thalassemia,Klinefelter Syndrome, Huntington Disease, autoimmune diseases,lipidosis, obesity defects, hemophilia, inborn errors of metabolism, anddiabetes.

Cancers which can be detected and/or determined by the process of thepresent invention generally involve oncogenes, tumor suppressor genes,or genes involved in DNA amplification, replication, recombination, orrepair. Examples of these include, but are not limited to: BRCA1 gene,p53 gene, APC gene, Her2/Neu amplification, Bcr/Abl, K-ras gene, andhuman papillomavirus Types 16 and 18. Various aspects of the presentinvention can be used to identify amplifications, large deletions aswell as point mutations and small deletions/insertions of the abovegenes in the following common human cancers: leukemia, colon cancer,breast cancer, lung cancer, prostate cancer, brain tumors, centralnervous system tumors, bladder tumors, melanomas, liver cancer,osteosarcoma and other bone cancers, testicular and ovarian carcinomas,head and neck tumors, and cervical neoplasms.

In the area of environmental monitoring, the present invention can beused, for example, for detection, identification, and monitoring ofpathogenic and indigenous microorganisms in natural and engineeredecosystems and microcosms such as in municipal waste water purificationsystems and water reservoirs or in polluted areas undergoingbioremediation. It is also possible to detect plasmids containing genesthat can metabolize xenobiotics, to monitor specific targetmicroorganisms in population dynamic studies, or either to detect,identify, or monitor genetically modified microorganisms in theenvironment and in industrial plants.

The present invention can also be used in a variety of forensic areas,including, for example, for human identification for military personneland criminal investigation, paternity testing and family relationanalysis, HLA compatibility typing, and screening blood, sperm, ortransplantation organs for contamination.

In the food and feed industry, the present invention has a wide varietyof applications. For example, it can be used for identification andcharacterization of production organisms such as yeast for production ofbeer, wine, cheese, yoghurt, bread, etc. Another area of use is withregard to quality control and certification of products and processes(e.g., livestock, pasteurization, and meat processing) for contaminants.Other uses include the characterization of plants, bulbs, and seeds forbreeding purposes, identification of the presence of plant-specificpathogens, and detection and identification of veterinary infections.

EXAMPLES Example 1 Particles Synthesis

This example demonstrates that various particles can be synthesized foruse according to the present invention. Exemplary methods are describedin detail below.

Exemplary particle batches were synthesized in 38-μm tallpolydimethylsiloxane (PDMS) microfluidic channels with the stop-flowlithography method. For the 12-plex study, code and inert buffer regionswere polymerized from monomer solutions with 35% (v/v) poly(ethyleneglycol) diacrylate (MW=700 g/mol) (PEG-DA 700), 20% poly(ethyleneglycol) (MW=200 g/mol) (PEG 200), 40% 3×Tris-EDTA (TE) buffer (pH 8.0),and 5% Darocur 1173 photoinitiator. 1× TE and rhodamine-acrylate (1mg/ml) were added to code monomer to give final concentrations of 9.4%and 0.6%, respectively. 1× TE and blue food coloring were added tobuffer monomer to give final concentrations of 8.0% and 2.0%,respectively. Food coloring was used to visualize stream widths. Proberegions were polymerized from a different monomer solution that wasadded to acrydite-modified DNA probe sequences (Integrated DNATechnologies, IDT) suspended in 1× TE to give the desired finalconcentration of probe, 18% (v/v) PEG-DA 700, 36% PEG 200, and 4.5%Darocur; the remaining balance consisted of 3× TE.

In an effort to coarsely rate-match the binding of the targets used inthis exemplary study, we incorporated the probe sequences at differentconcentrations in the particles (Table 1). As the characteristic timefor target depletion scales with the inverse square root of probeconcentration, a doubling of the binding rate for a given target willrequire a 4× increase in the amount of probe incorporated in a proberegion of fixed size. In this exemplary study all rates were adjusted tomatch that of let-7a binding. Without being bound to any particulartheory, it is contemplated that higher sensitivities and shorter assayscould have been achieved by loading probe at maximum concentration. Inthis particular case, the goal was to develop a 12-plex assay with broaddynamic range and ˜1 amol sensitivity for all targets.

TABLE 1Exemplary particle codes and probe information for batches synthesizedfor 12-plex study. Final composition (v/v) of PEG-DA 700, PEG 200, and Darocur 1173photoinitiator in prepolymer stream for probe were fixed at 18, 36, and 4.5%,respectively. Hairpin melting temperatures are listed in descending order, as calculatedfor the DNA-RNA duplex by IDT′s OligoCalc application for the incubation conditions usedin this exemplary study. For each miRNA, the relative binding rate was calculated usingthe average of target signals from 30- and 60-min incubations with 500 amol of targetand ligation labeling. Short incubations were chosen to ensure the system had notreached equilibrium. Quoted probe concentrations refer to prepolymer stream composition.Approximately 11% of the probe in the prepolymer stream was covalently incorporated intothe particles (Pregibon, D.C. et al., Anal. Chem. 81, 4873-4881 (2009)).Binding Rate Relative to Adjusted Hairpin let-7a for Probe Melting Probe Conc. Concentration Identifying Target Probe Sequence Temps (°C.) of 50 μM (uM) Codes let-7a 5Acryd/GAT ATA TTT TAA 38.0 1.00 5031131, ACT ATA CAA CCT ACT 32231, ACC TCA/3InvdT 31231, (SEQ ID NO: 1)32131, 31031 miR-21 5Acryd/GAT ATA TTT TAT 60.4, 52.9, 0.45 247 31112,CAA CAT CAG TCT GAT 49.3, 46.6 32212, AAG CTA/3InvdT 31212(SEQ ID NO: 2) 32112, 31312 miR- 5Acryd/GAT ATA TTT TAA 45.9, 44.7, 0.631.26 31132, 29b-2 ACA CTG ATT TCA AAT 41.7 32232, GGT GCT A/3InvdT 31232(SEQ ID NO: 3)  32132, 31032 miR-181b-1 5Acryd/GAT ATA TTT TAA58.4, 43.2, 0.89 63 32230, CCC ACC GAC AGC AAT 38.6 31130,GAA TGT T/3InvdT 31230 (SEQ ID NO: 4) 32130, 31030 miR-1435Acryd/GAT ATA TTT TAG 55.2, 51.1 1.04 50 31110, AGC TAC AGT GCT TCA32210, TCT CA/3InvdT 31210, (SEQ ID NO: 5) 32110, 31310 miR-1455Acryd/GAT ATA TTT TAA 47.4, 43.0, 1.01 50 31121, GGG ATT CCT GGG AAA36.6 32221, ACT GGA C/3InvdT 31221 (SEQ ID NO: 6) 32121, 31321 miR-146a5Acryd/GAT ATA TTT TAA 64.6, 49.4, 0.67 111 3001, ACC CAT GGA ATT CAG48.3, 43.4 31101, TTC TCA/3InvdT 32201 (SEQ ID NO: 7) 31201, 32101miR-210 5Acryd/GAT ATA TTT TAT 68.4, 65.6, 0.90 62 31122,CAG CCG CTG TCA CAC 59.7, 55.4 32222, GCA CAG/3InvdT 31222(SEQ ID NO: 8) 32122, 31322 miR-221 5Acryd/GAT ATA TTT TAG 49.8, 43.6,0.82 62 31111, AAA CCC AGC AGA CAA 42.2, 41.0 32211, TGT AGC T/3InvdT31211 (SEQ ID NO: 9) 32111, 31311 miR-222 5Acryd/GAT ATA TTT TAA68.2, 68.1, 0.62 130 31120, CCC AGT AGC CAG ATG 58.1, 47.5 32220,TAG CT/3InvdT 31220 (SEQ ID NO: 10) 32120, 31320 miSpike5Acryd/GAT ATA TTT TAA 66.5, 46.0 1.21 35 30002, GAC CGC TCC GCC ATC31102, CTG AG/3InvdT 32202 (SEQ ID NO: 11) 31202, 32102 RNU6B5Acryd/GAT ATA TTT TAA 64.4, 58.4, 0.95 55 30000, AAA ATA TGG AAC GCT56.9, 56.1 31100, TCA CGA ATT TGC GTG 32200 TCA TCC TTG CG/3InvdT 31200,(SEQ ID NO: 12) 32100

Code, buffer, and probe prepolymer solutions were loaded into four-inletmicrofluidic synthesis channels using modified pipette tips(Biosciences) as delivery chambers and forcing pressures of 4.5 psi.Hydrogel microparticles (250×70×35 μm) were simultaneously synthesized,encoded, and functionalized at rates up to 16,000 per hour with 100-msUV exposures (Lumen 200 at 75% setting, Prior Scientific) controlled bya shutter system (Uniblitz, Vincent Associates) interfaced with acustom-written Python automation script. Stream widths were adjustedsuch that code and probe regions spanned 140 and 40 μm, respectively, ofthe length of the particles. Buffer regions accounted for the remaining70 μm of the length. We also showed that the same particle dimensionscan easily accommodate two probe strips, with no loss in performanceupon incubation, labeling, and scanning.

Following polymerization, particles were flushed down the synthesischannel and collected in a 1.7-ml Eppendorf tube containing 950 μl ofTET (1× TE with 0.05% (v/v) Tween-20 surfactant (Sigma Aldrich)). Tweenwas added to prevent particle aggregation. Particles were next suspendedin 200 μl of PEG 200 for 5 min and then rinsed with 700 μl of TET. Thiswashing sequence was used to rinse the particles of unreacted PEG-DA,probe, and rhodamine. The wash sequence was repeated two more times andinvolved manual aspiration of supernatant facilitated by centrifugalseparation of the dense particles. Particles were stored in TET at finalconcentrations of ˜12.5 particles/μl in a refrigerator (4° C.).

Example 2 miRNA Incubation Experiments

This Example demonstrates typical sample incubation steps suitable foruse in the present invention.

For all exemplary incubations studied, particles synthesized, forexample, by the methods described in Example 1, were brought to roomtemperature prior to use, and each incubation was carried out in a totalvolume of 50 μl in a 0.65-ml Eppendorf tube with a final saltconcentration of 350 mM NaCl and all twelve types of particle present(˜360 particles/incubation tube). For calibration and specificitystudies, a hybridization buffer (TET with assay-specific NaCl molarity)was first added to the Eppendorf tube, followed by all relevant targetsequences (IDT) diluted in a mixture of 1× TE with 500 mM NaCl. Tweenwas excluded from the dilution buffer to prevent inaccuracies inpipetting steps that can arise from surfactant-induced changes inwettability. Depending on the assay type, either 1 μl of TET or 1 μl ofE. coli total RNA (200 ng/μl) was introduced. For tissue profilingstudies, hybridization buffer was added directly to a tube containingeither 2.5 or 1.0 μl of previously frozen extracted total RNA (oneindividual per tissue type; stored at 100 ng/μl). Primary pair samplesconsisted of total RNA isolated from primary tumor and its adjacentnormal tissue. Total RNA for all tissues was isolated by TRIzolpurification; integrity of isolation was confirmed by checking forintact 18S and 28S ribosomal RNA. Lung sample (BioChain) was obtainedfrom 50-year-old male with poorly differentiated squamous cellcarcinoma. Breast sample (BioChain) was obtained from 53-year-old femalewith moderately differentiated invasive lobular carcinoma. Stomachsample (BioChain) was obtained from 70-year-old female with poorlydifferentiated adenocarcinoma. Pancreas sample (BioServe) was obtainedfrom 65-year-old female with well-differentiated acina cell carcinoma.For all exemplary assays, 1 μl of miSpike (IDT) appropriately diluted in1× TE with 500 mM NaCl was also introduced to give a total amount of 100amol of the synthetic sequence to measure consistency ofscanning/labeling and for quantification purposes. Prior to the additionof particles, incubation mixtures were heated to 95° C. for 5 min in aMulti-therm shaker (Biomega) and then brought back to room temperatureover a 7 min period. A previously prepared master mix of particles (18per μl) was thoroughly vortexed for 1 min, and 20 μl (˜30 particles ofeach probe type) was introduced to each incubation tube. Incubation withtarget was carried out at 55° C. for 90 min in a thermomixer (QuantifoilRio) with a mixing speed of 1800 rpm.

Following hybridization with target, samples were rinsed three timeswith a solution of 500 μl TET containing 50 mM NaCl. Supernatant wasmanually aspirated from the tube following centrifugal separation of theparticles. All but 50 μl of solution was aspirated after the thirdrinse. Next, 245 μl of a previously prepared ligation master mix (100 μl10× NEBuffer 2, 875 μl TET, 25 μl of XXXATPcarrier, 250 pmol of ATP, 40pmol of universal adapter, and 800 U of T4 DNA ligase) was added to thetube. The mixture was placed in the Multi-therm shaker at 21.5° C. for30 min with a mixing speed of 1500 rpm. Following ligation, an identicalthree-rinse cycle was performed. Streptavidin-r-phycoerythrin reporter(SA-PE, 1 mg/ml) was diluted 1:50 in TET and added to obtain a finaldilution of 1:500. Samples were incubated in the Multi-therm unit at21.5° C. for 45 min. After another three-rinse cycle, particles wereadditionally rinsed in 500 μl of PTET (5× TE with 25% (v/v) PEG 400 and0.05% Tween-20), and then suspended in a final volume of 50 μl PTET forscanning. Prior to use, all PTET was sonicated for 5 min to eliminateaggregations of polymer.

Example 3 Detection Using Multifunctional Particles

In this Example, hydrogel particles were use. The synthesis ofchemically geometrically complex hydrogel microparticles can be carriedout using the flow lithography technique explained in detail in U.S.Pat. No. 7,709,544.

By polymerizing across laminar co-flowing streams of monomer,multifunctional particles with distinct chemical regions can be rapidly(>10⁴/hr) produced with high degrees of reproducibility. Separate “code”and “probe” regions are used to identify particles and capture targets,respectively. The bulk-immobilization of probe molecules in thebio-inert, PEG-based gel scaffolds provides solution-like capturekinetics and high degrees of both specificity and sensitivity, leadingto significant advantages over surface-based immobilization strategiesemployed in microarrays and existing particle systems. Patterns ofunpolymerized holes in the code portion of the particle serve as thebasis for a graphical multiplexing barcode to identify the probe(s) in aparticular particle. Unlike bead-based systems that use optical encodingof spheres, an arrangement in which particles have multiple distinctregions makes single-color scanning possible, with only one excitationsource and one detector required (FIG. 8a ). Furthermore, the codinglibrary can easily be expanded to accommodate high levels ofmultiplexing or parallel processing of samples. Other methods forencoding these particles can also be implemented as discussed above; forinstance, the particles can bear stripes with variable fluorescentintensities (of one or multiple wavelengths), optical properties,dimensions, etc.

In addition to bearing a code, particles also bear a probe region wheretargets are captured for quantification. The probes typically consist ofspecies of biomolecules that bind specifically to a target of interest.For nucleic acid detection, probes typically consist of DNAoligonucleoties. A suitable DNA probe design and labeling methodologycan be employed for a post-hybridization labeling method thataccommodates operation of a gel particle scanning system forhigh-throughput multiplexed miRNA quantification. In the discussionbelow, particle synthesis, incubation, and scanning steps are describedin detail for miRNA quantification, but this is provided as one exampleonly, and it is to be recognized that such techniques are applicable tonucleic acids in general and are herein contemplated.

It is possible to use encoded particles with a post-hybridizationlabeling scheme and a suitable scanner, e.g., a slit-scan system, toperform rapid, multiplexed analysis of nucleic acids (FIG. 8b and c ).

In some embodiments, particles are designed to be scanned rapidly in aflow-through system such that the fluorescent signal obtained along eachparticle is integrated across the particle width by the detector. Theparticles each have a fluorescent code-region, bearing a series of holesthat are used to identify the particle, negative control regions, and atleast one probe region where targets are captured and labeled. The sizesof the holes in the code region determine the depths of the fluorescencetroughs in the signature and thus indicate the particle identity. Weoptimized the particle architecture and hole design (FIG. 9) to findthat four distinguishable coding levels (0-3) could be obtained for70-μm wide particles, leading to 192 possible codes for a five-barparticle (FIG. 8c ). Multiplexing capacity could easily be augmented to>10⁵ by adding more bars, using multiple fluorescent levels for the coderegion, or incorporating multiple probes on each particle. We developedand trained a decoding algorithm written in MATLAB to accurately decodeparticles and quantify targets. Any suitable decoding technique can beemployed. In the example algorithm, particle orientation (code- orprobe-first) and velocity are determined to analyze the coding holes andestablish a first estimate of code identity. A revised assignment iscalculated by checking the consistency among holes identified as thesame level, and a decoding confidence score is then computed and used toaccept or reject particles. This method typically provides a decodingaccuracy of ˜98%, with only ˜10% score-based rejection at throughputs upto 20 particles/s.

Example 4 Post-Hybridization Labeling

To generate a detectable signal indicating the presence and capture ofnucleic acid targets, an exemplary post-hybridization ligation-basedmethodology is provided and demonstrated in this Example and Example 5for labeling.

Such a post-hybridization method can be used to fluorescently labelbound selected targets, e.g., miRNA targets. Existing approaches rely onthe bulk-labeling of RNA using chemical or enzymatic means. Thesemethods may suffer from high cost, the need for small-RNA purificationand clean-up, sequence bias due to secondary structure, or complicated,time-consuming protocols. Here, we provide, for example, a two-stepmethod to efficiently label targets after hybridization in about onehour.

Experimentally, we used T4 DNA ligase to link a universaloligonucleotide adapter to the 3′ end of targets captured ongel-embedded DNA probes that act as a ligation templates (FIG. 10). Assuch, we can use a common, universal adapter to label multiple targetsin a single reaction. The labeling process requires only a few simplesteps. First, particles are hybridized with the sample, in this casetotal RNA, to capture appropriate targets in the particle probe regions.After excess sample is rinsed away, a ligation mix is added thatincludes the appropriate enzymes, all important co-factors (such asATP), and a common biotinylated adapter. After a short reaction(typically 5-60 min) at room temperature, a low-salt buffer is used torinse away any unreacted adapter. After rinsing away unreacted adapter,the particles are incubated with phycoerythrin-conjugated streptavidinreporter (SA-PE) to provide fluorescence. After another rinse, theparticles can then be analyzed. More importantly, this labeling methodwas very efficient, had no minimal input RNA requirement, and showed nosequence bias for the targets used in this exemplary study (Examples 4and 5). For each new miRNA target species, we incorporated atarget-specific sequence into the universal probe template; complexmodification and customization were not necessary.

In this arrangement, the adapter sequence was designed to minimize probehairpin formation, which could retard target hybridization, and providean adapter-probe melting temperature T., that was ˜10-20° C. in ligationbuffer. Although we used a reduced salt buffer during the rinse, thedehybridization of unreacted adapter can be accomplished using anycondition that destabilizes nucleic acid interactions (low salt, hightemperature, additives such as DMSO, PEG, or glycerol, etc.). Typically,we use SA-PE reporter to achieve maximum fluorescent signal. In additionor alternatively, a ligation-based labeling can be performed withadapters that are directly labeled with fluorophores or other reportingentities. Without being bound to any particular theory, it would beappreciated that this reduces the time and complexity of the assay. Theprocess can be used, with appropriate probe and adapter design, toligate adapters to the 3′ end of DNA or RNA species containing a 3′ OH,or at the 5′ end of these species containing a 5′ phosphorylation.

Example 5 Optimization and Variations of Ligation-Based Labeling

In various embodiments, several aspects of the labeling techniquedescribed in the present invention were optimized, includingprobe/adapter design, reagent concentrations, rinse buffer salt content,ligation time, and ligation temperature. We show here the effects ofligation time and adapter tail length on labeling efficiency. Thenucleic acid probes, targets, and adapters (all received from IntegratedDNA Technologies, IDT) are given in the table below.

TABLE 2Nucleic acid probes and targets used in optimization studies. Sequencein bold represents universal adapter-specific sequences, sequence in regularrepresents target-specific sequences, and sequence underlined representspoly(A) tails. Oligo Name: Sequence/Modifications: let-7a probe, DNA/5Acryd/GATATATTTTAAACTATACAACCTACTACCTCA/3InvdT/ (SEQ ID NO: 13)let-7a target, RNA 5′-UGAGGUAGUAGGUUGUAUAGUU-3′ (SEQ ID NO: 14)UA10-Cy3, DNA /5Phos/TAAAATATAT/3Cy3/ (SEQ ID NO: 15) UA10-bio, DNA/5Phos/TAAAATATAT/3Bio/ [poly(A) = 0] (SEQ ID NO: 16) /5Phos/TAAAATATATAAA/3Bio/ [poly(A) = 3] (SEQ ID NO: 17) /5Phos/TAAAATATATAAAAAA/3Bio/ [poly(A) = 6] (SEQ ID NO: 18) /5Phos/TAAAATATATAAAAAAAAAAAA/3Bio/ [poly(A) = 12] (SEQ ID NO: 19)

Adapter/Probe Design

Exemplary probes described above were designed to include amiRNA-specific region and an adapter-specific region, such that whenbound, the 3′ end of the miRNA target would abut the 5′ end of theadapter. We chose to label the 3′ end of miRNA targets because it hasbeen demonstrated that when using a DNA template, the action of T4 DNAligase in joining DNA to RNA molecules proceeds several orders ofmagnitude more rapidly at the 3′ end of RNA versus the 5′ end (Bullard,D. R. et al., Biochem J 398, 135-144 (2006)). The adapter sequence andlength were chosen such that (1) the melting temperature was between10-20 C in ligation buffer, (2) the sequence was not significantlyself-complementary in order to avoid adapter hairpin or homodimerformation, and (3) complete DNA probes (with adapter and miRNAsequences) did not show appreciable hairpins for the miRNAsinvestigated.

Ligation Time

We performed studies to determine the minimum ligation time needed forour labeling assay, using let-7a as a model system. Particles bearing alet-7a DNA probe region were incubated with 5 fmol synthetic let-7a RNAat 55 C for 110 min. Particles were rinsed three times with phosphatebuffered saline containing 0.05% Tween-20 (PBST, pH 7.4, Fluka) andincubated with 250 □l of a ligation mix containing 200 U T4 DNA ligase,40 nM Cy3-modified adapter (UA10-Cy3), and 0.05% Tween-20 in T4 DNAligation buffer (NEB) for 10, 30, or 90 min at 16 C. After ligation,particles were rinsed three times in TE containing 0.025 M NaCl,deposited on a glass slide, and imaged using a CMOS camera (ImagingSource). We measured the fluorescence intensity in the probe region ofeach particle, subtracting the background fluorescence to get a targetsignal, which indicated ligation efficiency. The results are shown inFIG. 11.

We calculated the relative efficiency by normalizing each signal by thatobtained for the 90 min sample. As can be seen in FIG. 11, ligationis >95% complete even after a short 10-min reaction. For the experimentsdescribed in this work, we chose to use a ligation time of 30 min toensure nearly complete ligation.

Tail Length for Biotinylated Adapters

The reporter streptavidin-phycoerythrin (SA-PE) is a large proteinstructure that has a radius of gyration on the order of ˜10-15 nm. Assuch, when using biotinylated adapters with the SA-PE reporter, we foundthat it was beneficial to extend the biotin group away from the polymerbackbone of the gel matrix. To do this, we used a poly(A) tail at the 3′end of the adapter and investigated the effect of tail length on targetsignal.

In this experiment, we used the same let-7a particles as in the previoussection. We incubated with 50 amol let-7a miRNA for 60 min at 50 C. Theparticles were rinsed three times in PBST, and divided into fourseparate tubes. Particles in each tube were incubated for 30 min at roomtemperature with ligation mix containing 200 U T4 DNA ligase, and 40 nMUA10-bio (with either a 0, 3, 6, or 12 bp poly(A) tail), in 1×-T4 DNAligation buffer (NEB) with 0.05% Tween-20. After ligation, particleswere rinsed three times in TE containing 0.05 M NaCl and 0.05% Tween-20.Particles were deposited on a glass slide and imaged using an EB-CCDcamera. The target signals were compared to determine the effect ofpoly(A) tail length, as shown in FIG. 12.

As can be seen in FIG. 12, the length of the poly(A) tail has a largeeffect on target signal obtained. From zero to 12 bp, the signalincreases ˜5× but seems to level off at that point. For the experimentsdescribed in some examples, we chose to use universal adapters withpoly(A) tail lengths of 12 bp.

In various embodiments, a wide range of alternative techniques andsystems to those described above can be successfully employed. Examplesof such are provided here.

Direct Adapter-Based Labeling Using Fluorophore-Conjugated Adapters

Instead of using a technique in which biotinylated adapters are ligatedand later reported with streptavidin-conjugated fluorophores,fluorophores can be used directly. When ligating to the 3′ end ofhybridized targets, the universal adapters will have desired afluorophore incorporated, preferably at the 3′ end or on one of theinternal nucleotides. As illustrated in Example 13, this methodeliminates one step in the process, making it more simple and rapid.

Multiplexed Detection Using Adapters with Different Fluorophores

For some applications, it can be important to detect multiple nucleicacid species in a common region. When the probes are not separated indistinct regions of a particle or substrate, it is possible to performmultiplexed detection using adapters modified with fluorophores thathave unique emission spectra. For example, three probes that each have aunique adapter probe sequence can be used in one region with adaptersmodified with 3 unique flourophores. An example of this is shown in FIG.14.

Alternately, for some applications it can be important to detectvariability at the end of a target (e.g., targets with nucleotidescropped from one end). In this case, a similar probe can be used, butmultiple adapters (preferably with different fluorohpores) are used thatextended a different number of nucleotides into the target probe region.Ligation would only occur if the target/adapter ends perfectly abut,thus the target end sequence(s) can be determined by measuring thelevels of each fluorophore used for the various adapters. Alternately,adapters bearing the same fluorophore may be used with two separatequantification steps run in parallel (with two samples) or series (samesample but two ligation steps).

In the case of both labeling and universal encoding, ligation can beachieved at the 5′ or 3′ end of the adapters, especially when allspecies involved are DNA. When using DNA Ligase, it is known thatligation is much more efficient at the 3′ end of RNA targets (i.e., the5′ end of the DNA adapter). Adapters may be DNA, RNA, or any type ofnucleic acid analog. The nucleotides in the adapters or probes may bemodified as locked nucleic acids, or otherwise.

Use of Other Functional Adapters

Fluorophores were employed for encoding and labeling in the experimentsdescribed above, but it is understood that other types of functionalspecies can also be used, including but not limited to: chromophores,radioactive species, magnetic materials, quantum dots, etc. It is alsounderstood that universal encoding can be achieved using an adapterbearing an intermediary species (e.g. biotin), and functionalization(e.g. fluorescence) can be added in an additional step. Adapters canhave fluorophres at the end of their structure or along their backbone(e.g. fluorescent nucleotides). Another approach is to use intercalatingDNA/RNA dyes (like PicoGreen, YOYO-1, etc.) to introduce fluorescence inuniversal encoding or labeling. These may be used in conjunction withenzymes like exonuclease that will selectively degrade nucleic acidspecies that are not protected from digestion. In this scenario,adapters with longer sequences or more secondary structure will lead tobrighter signals from the intercalating dyes. In a different scenario,adapters may also bear specific nucleic acid sequences (tags) that canbe targeted in subsequent processing to add fluorescence (e.g., usingfluorophore-conjugated complementary oligonucleotides).

Rinse Free Labeling

It is possible to use the ligation-based labeling technique for analysisof particles without rinsing. In one example, ligation is carried out ata lower temperature (e.g., below the melting temperature, Tm, of theadapter) than scanning/analysis (which can be done above the Tm of theadapter). The melting temperature of the adapter can be adjusted viasequence, salt concentration, locked nucleic acids, etc. to denaturefrom the probe template at temperatures below, near, or above thetemperature used when analyzing particles. Ligation and scanning can beperformed right at or slightly above the Tm of the adapter—this stillallows ligation (likely with decreased efficiency) with minimal residualadapter bound to the probes during analysis.

Example 6 Particle Scanning

Typical scanning methods suitable for use in the present invention aredescribed in this Example.

Focusing devices (35 μm in height) with two inlets, one outlet, fourside streams, and a 125-μm wide detection region were mounted on a ZeissAxio Observer microscope equipped with a Zeiss Plan Neofluar 20×objective (NA 0.50) (FIG. 9a ) (Chapin, S. C., et al., Lab Chip 9,3100-3109 (2009)). A chrome-coated soda-lime glass mask (AdvanceReproductions) was fitted into an iris slider bar and inserted into thefield stop of the microscope to limit the beam spot of a 100-mW, 532-nmlaser (Dragon Lasers) to a thin excitation window of 4×90 μm in thescanning plane. Prior to each scanning session, laser alignment wascalibrated with a power meter (Newport, Model 1815-C). Using imagescaptured from a Clara Interline CCD camera (Andor Technology), theexcitation window was oriented such that its long dimension was alignedperpendicular to the flow direction approximately 750 μm from the exitport of the device. A switching box on the side port of the microscopewas used to alternate between the CCD and a photomultiplier tube (PMT,Hamamatsu H7422-40) used to record fluorescence signatures of passingparticles.

PTET was injected from a reservoir input to serve as a focusing sheathstream. For each trial, particle-bearing fluid was aspirated into amodified pipette tip using a syringe connected to the tip via Tygontubing. The tip was inserted into the appropriate PDMS inlet port and apressure of 8 psi was used to drive the flow of both fluids. A typicalscan of 50 μl of particle-bearing fluid lasted ˜30 s and used less than25 μl of sheath fluid. Particle throughputs ranged from 5-25 per second,depending on the number of particles used in the assay. Devices wereable to be used more than 50 times without degradation. Following eachscan, a rinse solution of 30 μl 1× TE was flowed through the particleinlet to flush out stranded particles and thereby reduce inter-runcontamination. Additionally, the loading tip was rinsed in ethanol andwater so that it could be reused. With manual loading from Eppendorftubes, eight samples could be scanned and analyzed in 30 min, leading toa projected throughput of ˜125 samples per 8-h workday. In futureapplications of this technology, automation of the particle-loading andrinsing processes using well-plates and a computerized liquid handlingsystem will greatly augment efficiency (>500 samples/day).

The output current of the PMT was conditioned using a homemade amplifierwith a low-pass filter, and the resulting voltage signal was captured ata rate of 600 kHz by a digital acquisition (DAQ) board (USB-6251,National Instruments). A Python script was written to convert each scanto a binary text file for off-line analysis. Single-chemistry particleswith fluorescent rhodamine incorporated throughout were scanned tooptimize the performance of the scanning system, leading to acombination of amplifier gain (22), cutoff frequency (100 kHz), slitwidth (4 μm), and PMT control voltage (0.300 V) that produced thehighest signal-to-noise ratio (SNR) and frequency response possible.Furthermore, by scanning particles with various barcode designs, it wasobserved that a minimum spacing of 8 μm was required between holes toprevent mechanical deformations of the soft hydrogels during flowalignment. The four-level code design was employed based on studies thatsystematically varied the size of the holes to determine effects ontrough depth in scan profiles (FIG. 9b and c ).

Example 7 Data Analysis

Typical data analysis in accordance with the present invention aredescribed in this Example.

Raw data files (20 million points/scan) produced by the scanning processwere analyzed with a custom written MATLAB algorithm designed to isolateindividual particle signatures, identify the code displayed by eachparticle, and quantify the amount of target bound. The algorithmprocessed scans of 50-μl samples in under 5 s, making the approachsuitable for high-throughput applications. In the initial filter step,the algorithm excised portions of the scan that exceeded a thresholdvoltage and then interrogated each removed segment for characteristicsthat identified it as a particle signature. Using specific properties ofthe fluorescent code region as reference points, a high-confidenceestimate of the velocity of each particle was determined and utilized topinpoint trough locations for the five coding holes. The orientation ofthe particle (i.e., probe- or code-first) was established using thefixed-value “3” hole that bordered the inert buffer region. After aninitial code identity was calculated from the trough depths, a secondaryreview was conducted by measuring the standard deviation in troughdepths of holes designated to be of the same level and corrective actionwas taken if necessary. In the final decoding step, a confidence scorewas calculated for the particle by computing the linearity of thecorrelation between trough depth and assigned level. A particle decodingevent was rejected if its Pearson coefficient fell below 0.97.

In order to calculate the amount of target bound, the measured particlevelocity was used to infer the location of the center of the proberegion. Briefly, a search window was used to investigate the scan inthis region, seeking to identify a local maximum that could becorrelated to a target-binding event. If a maximum was found, theposition of the search window along the scan profile was adjusted untilthe two endpoints were sufficiently close in signal amplitude, therebyselecting a nearly symmetrical portion of the maximum over which toaverage for quantification purposes. In the cases in which a maximum wasnot found, the original estimate of probe center was used to calculate amean signal without a search window. To calculate the background for agiven probe sequence and incubation condition, particles from the samesynthesis batch were incubated in the presence of only 100 amol ofmiSpike target according to the procedure described above. This methodprovided a measure of the probe-dependent background that arose from thePEG scaffold and the universal adapter used in the labeling process.Also, upon calculation of all code identities and target levels, aparticle would be rejected from consideration if its target level wasmore than one inter-quartile range above the third quartile or below thefirst quartile of the data set consisting of target levels associatedwith the probe in question. This measure was taken as further protectionagainst incorrect code assignments and inter-run contamination.

For calibration and profiling studies, mean background-subtractedsignals were computed for each target at each incubation condition. Forinter-run comparisons of calibration data, signals were normalized bybackground-subtracted miSpike amplitude, with the null (0 amol) samplesproviding the reference 100-amol miSpike value for both neat and E. coliinvestigations. miSpike target values displayed on the calibrationcurves (FIG. 15 and FIG. 16) were not adjusted to this reference inorder to demonstrate the repeatibility of the labeling and scanningprocess. For profiling studies, the background-subtracted miSpike signalfrom the first scan of each healthy tissue type was used as thereference for analysis of that tissue. Signals from a given profilingscan were further normalized by the RNU6B amount in that scan tofacilitate direct quantitative comparisons that were independent oftotal RNA amount. Repeat runs of tissue assays were conducted at leastone day after the original. For each calculated expression ratio, thehealthy and tumor samples were assayed and scanned in the same set ofexperiments for consistency. We required at least 2 amol of target to bedetectable in a tissue of a given disease state in order to calculate anexpression ratio. As we only used a single patient sample for eachtissue type, we implemented a threshold approach to determinedysregulation. For each target in each tissue, the three log-transformedexpression ratios from the three separate trials were used to calculatean SNR, by dividing the mean of the set by the standard deviation.Targets with SNRs above 3 were considered to be dysregulated. It shouldbe noted that all 20 instances of dysregulation were able to becorrelated to observations from the literature regarding the expressionprofiles of either mature miRNA (16 of 20) or miRNA precursors (4 of20).

Example 8 miRNA Profiling

The experiment described in this example demonstrates that compositionsand methods provided in the present invention may be use for variousapplications (e.g., miRNA profiling).

Experimentally, this technique was proven by an investigation into thedynamic range, sensitivity, and specificity of the platform in thecontext of a 12-plex assay featuring ten clinically relevant miRNAtargets. Because of its relative invariance across tissue types anddisease states, RNU6B was used as an internal control for normalizationpurposes. We also used 100 amol of miSpike (a synthetic 21-mer) as anexternal control to validate the consistency of the labeling andscanning processes. We synthesized twelve batches of single-probeparticles for this study. To compensate for discrepancies in targethybridization rates, we implemented a coarse rate-matching by tuning theprobe concentration for each target using previously determined scalinglaws (Table 1). To fully demonstrate the versatility of the scanner,five separate codes were correlated to particles of each probe type,thereby simulating a 60-plex assay.

To further assess the sensitivity and dynamic range of our system, wesimultaneously spiked four of the twelve targets into 50-μl incubationmixes at amounts ranging from 1 to 2187 amol. We observed a lineardetector response over four logs, with sub-attomole sensitivity achievedfor three of the four targets and strong agreement between neat samplesand those spiked with 200 ng of E. coli total RNA to add complexity(FIG. 15 and FIG. 16). By comparison, existing bead-based approacheshave a 200-amol limit of detection and only one log of range. To assessspecificity, we performed assays with let-7a particles and four membersof the let-7 family spiked separately at 200 amol into samplescontaining 200 ng E. coli total RNA. Scans revealed a maximumcross-reactivity of 27% (FIG. 15b ), which is lower than other systems(microarray ˜50%) and can be dramatically improved with lowerhybridization salt concentrations (FIG. 17) These assays were veryreproducible, with intra- and inter-run COV' s of 2-7% (Table 3). Due tolimitations in detection and particle preparation, it is common forusers of current bead-based systems to employ 4,500 copies of each typeof bead in an assay for high-confidence estimates of target level. Bycontrast, we found it sufficient to analyze only 10-15 hydrogelparticles for each probe type (FIG. 18).

TABLE 3 Intra-run COVs in target level for E. coli calibration curve.All entries are percentages with each statistic calculated using 19particles on average. miR-222 exhibited a limit of detection over 1amol. Inter-run COV in background-subtracted miSpike signal (100 amol)for the nine represented scan sets was 6.84%. 1 3 5 9 27 81 243 729 2187Target amol amol amol amol amol amol amol amol amol miR-210 59.45 29.2210.88 10.93 1.81 5.91 1.39 5.85 1.93 miR-221 36.71 9.95 21.80 18.41 4.117.20 2.79 6.81 2.01 miR-222 — 5.96 16.10 15.62 4.85 5.25 3.26 5.93 3.27let-7a 87.99 19.18 26.77 18.83 5.20 5.83 3.13 5.53 2.93

As a further validation of the platform, we performed expressionprofiling across tumor and adjacent normal tissue for several cancertypes. As anticipated, we observed the dysregulation of several miRNAtargets in all of the diseases investigated (FIG. 15c , Table 4, andTable 5). Although we used 250 ng of total RNA for these samples,similar results were obtained for lung samples using only 100 ng,suggesting that less input RNA would be sufficient. With a total assaytime of only 3 h, the profiling is more efficient than microarrayapproaches (˜24 h) and exhibits sensitivity and reproducibility farsuperior to that of existing bead-based methods.

TABLE 4 Mean target amounts and inter-run COVs in target amount for250-ng tissue profiling replicates. Top number in each entry is meanamount for replicate trials (amol); bottom number in parentheses is theinter-run COV (%). Amounts were determined by comparison to thebackground-subtracted 100-amol miSpike signal from each run. Replicateassays were conducted on different days to rigorously testreproducibility. Each statistic was calculated using 16 particles onaverage. Entry spots lacking data indicate that target was not presentabove the 2 amol cutoff. miR- miR- miR- miR- miR- miR- miR- miR- miR-let-7a 21 29b-2 181b-1 143 145 146a 210 221 222 RNU6B Lung 594.811498.85 68.36 7.02 85.61 162.88 77.02 — 7.21 8.13 57.16 Tumor (3.18)(10.57) (10.52) (21.45) (10.55) (6.05) (8.28) — (8.09) (8.22) (7.46)Lung 368.08 141.70 31.80 2.69 59.79 189.07 6.95 — — 6.45 10.84 Healthy(13.97) (12.10) (8.43) (18.61) (10.54) (8.98) (12.61) — — (12.33)(10.65) Breast 1094.19 808.08 65.88 3.71 32.48 73.53 9.26 — — 2.29116.88 Tumor (6.82) (9.96) (9.99) (5.01) (7.48) (11.20) (4.75) — —(16.14) (0.19) Breast 912.95 302.39 32.87 2.65 59.01 149.06 12.90 — —10.20 78.55 Healthy (6.30) (5.81) (25.08) (6.96) (8.52) (9.57) (5.89) —— (2.59) (3.05) Stomach 270.64 561.87 68.78 2.28 169.45 388.39 29.66 —2.93 14.15 175.69 Tumor (8.96) (13.76) (19.21) (11.03) (3.99) (9.16)(2.89) — (22.98) (8.16) (8.52) Stomach 258.28 204.24 73.44 — 186.39597.31 3.33 — — 7.83 78.45 Healthy (4.62) (24.90) (1.78) — (16.68)(17.91) (10.20) — — (5.76) (9.26) Pancreas 44.96 14.96 9.95 — 5.43 22.63— — — — 9.49 Tumor (2.62) (12.21) (17.82) — (58.64) (11.60) — — — —(8.23) Pancreas 98.10 18.21 14.85 — 6.79 10.88 — — — — 10.33 Healthy(2.64) (48.23) (11.77) — (27.57) (7.42) — — — — (7.81)

TABLE 5 Log-transformed expression ratios for 250-ng assays. Top numberin each entry is the mean of the log-transformed ratios of tumoramount-to-healthy amount of the indicated target in the specified tissueover three trials; bottom number in parentheses is the standarddeviation. Entry spots in red indicate dysregulation. Entry spotslacking data indicate that the ratio was not calculated. miR- miR- miR-miR- miR- miR- miR- let-7a 21 29b-2 143 145 146a 181b-1 222 Lung −0.51190.3020 −0.3911 −0.5670 −0.7870 0.3232 −0.3066 −0.6210 (0.0161) (0.0245)(0.0225) (0.0364) (0.0450) (0.0127) (0.0194) (0.0364) Breast −0.09420.2532 0.1378 −0.4318 −0.4801 −0.3168 −0.0266 −0.8253 (0.0349) (0.0416)(0.0777) (0.0137) (0.0194) (0.0433) (0.0529) (0.0591) Stomach −0.33100.0966 −0.3844 −0.3877 −0.5336 0.6007 0.3294 −0.0935 (0.0747) (0.0031)(0.1547) (0.0107) (0.0252) (0.0282) (0.3374) (0.0716) Pancreas −0.3023−0.0198 −0.1402 −0.1251 0.3534 — — 0.1785 (0.0345) (0.1183) (0.0778)(0.2381) (0.1019) — — (0.1382)

This high-performance nucleic acid profiling system and platform istherefore shown to employ a versatile scanning and labeling methodologythat enables the use of graphically-encoded hydrogel microparticles. Thesystem's unprecedented combination of sensitivity, flexibility, andthroughput offer exciting possibilities for discovery and clinicalapplications, particularly in the quantification of low-abundance miRNAand other biomolecules in readily-accessible media like serum.

Example 9 Scanning of Multiple-Event Particles

An example of how our approach is distinguished from standard cytometeryis shown in FIG. 19 below. In this example, the functional regions ofmultifunctional particles can be loaded with entities that cause scatterof the illumination, which in turn triggers the cytometer to record anevent.

We show a particle architecture that has two encoding regions and asingle probe region where target is captured. The two code regions havevarying levels of fluorophores embedded to give distinct signatures offluorescence in the three fluorescence channels. One code regions isintentionally wider than the other in order to indicate particleorientation. The target could be labeled with a fluorophore thatpreferentially appears in a single fluorescence channel, as shown. Inthis example, each particle would be reported as 3 events. Of thesethree, the first and last would give code information while the secondevent would be used for target quantification. In this manner, the codeand captured target are quantified non-contemporaneously.

We performed preliminary experiments to demonstrate the implementationof this methodology. We synthesized multifunctional particles that were˜200×35×30 μm with two fluorescent regions (30 μm and 60 μm, each dyedwith Cy5 and Cy3 fluorescent dyes) flanking a broad inert region. Theparticles were run through an Accuri C6 cytometer with a flow rate of100 μl/min and a core size of 40 μm. The threshold was set at 100,000 onFL4-H (which detects Cy5).

Each event recorded by the cytometer is given a timestamp with aresolution of 1 ms. As particles typically move at rates of ˜1 m/sthrough the flow cell, the interrogation of a particle that is 200 μmlong is expected to last ˜0.2 ms. As such, it can be expected that thetwo events recorded from a single multifunctional particle would appearin the same timestamp. To show that each particle was being read as twoseparate events, we plotted a histogram showing the count of timestampsthat had a given number of events. We would expect the number of eventsper timestamp to be even for our particles (2 events for a singleparticle, 4 events for two particles, etc.), and both odd and even forregular particles. As a control, we also ran standard Accuri 8-peakcalibration beads, with a typical spherical shape. The results are shownin FIG. 20.

As can be seen, the calibration beads are scanned fairly randomlythroughout the course of data acquisition, giving a range from 1-4beads/timestamp. The multifunctional particles, on the other hand, showclustering of 2 or 4 events per timestamp, which lends very well to thetheory that each particle is being read as two events. In addition, itcan be clearly seen from the plots of event vs. time that during eachtimestamp, there is a high- and low-level fluorescence reading. Theparticles were designed to have one bright and one dim region offluorescence in the FL-2 channel, which also gives support to the theorythat each particle is being read as two discrete events. This approachcan be applied to three or more events per particle as well. Eachregion/event can vary in terms of fluorescence level, forward or sidescatter, and width.

In some cases, it is useful to incorporate distinct levels of multiplefluorophores into each code region of the multifunctional particles. Asa proof-of-concept, we used rod-shaped particles, 200×35×30 μn, with asingle 60 μm code region on one end. The code region was labeled usingfour distinct levels of Cy3 and Cy5 fluorescent dyes. Particles wereanalyzed using the Accuri C6 cytometer with a flow rate of 100 μl/min, acore size of 40 μm, and a threshold of 5000 on FL4. The results areshown in FIG. 7 below.

The plot in FIG. 7 shows that it is possible to create a distinctfluorescent fingerprint in each code region of multifunctionalparticles. Each cluster of data points represents a distinct code.

For data analysis using this approach, an algorithm will be needed thatgroups events into particles, orients the particles, normalizesfluorescence against a standard if desired, and quantifies thefluorescence, scatter, or event width in each code and probe region. Thecorresponding code for each particle can then be given a confidencelevel, and those that were not called with a pre-defined level ofconfidence can be excluded from the analysis. The fluorescence in theprobe region can then be used to determine the amount of target presentin the sample analyzed. This system can be easily automated usingsoftware that performed analysis during or after scanning.

Example 10 Reading of Raw Signal

This Example demonstrates interrogating multifunctional particles instandard flow cytometers. In some embodiments, interrogation isperformed to acquire signal from a cytometer detector before it isprocessed into events by the machine's firmware and use custom softwareto identify, orient, and analyze particles scans. We performedproof-of-concept scanning of particles in this manner, using threeseparate cytometers from Partec, Accuri (C6), and Millipore (Guava).

To gather raw data, we used the leads (Partec and Millipore) or QC pin(Accuri) from a single PMT in each cytometer, connected them through asimple circuit (often just a single resistor), and measured the voltageusing a standard data acquisition (DAQ) board (National InstrumentsNIDAQ-USB6250). A custom script written in Python was used tocommunicate with the DAQ board, allowing the user to input how manysamples to acquire and at what frequency. Samples were taken at ratesranging from 60 kHz to 1 MHz. After acquisition, the data were stored ina single file.

For analysis, we applied Fast-Fourier-Transform-based filtering toisolate the desired frequency response for each scan. Then, particleswere identified in each sample by setting a threshold. If the signal wasfound to be above the threshold for a predefined number of samples, theregion of interest and its flanking data points were stored as a singleparticle scan. Design features built in to each particle were used toidentify code and probe regions. In addition, each signal could benormalized by a given feature on each particle. Our barcodes in thisexample consisted of series of stripes along the particle that hadvarying levels of fluorescence.

We used a standard set of test particles to assess alignment andconsistency of particle-to-particle scan in three commercial cytometers.We synthesized rod-shaped fluorescent particles bearing three distinctregions. Static image scans from regular fluorescence microscopy werecompared to those acquired from the raw scans obtained from a single PMTof each machine. After applying FFT-based filtering to isolate thedesired frequency response for each machine, the signal from eachparticle identified was scaled (x-axis only) to compensate forvariations in speed and plotted along a common x-axis. Typical resultsare shown in FIGS. 21 and FIG. 22 with overlain particle scans anddistribution of event width (which inversely correlates with particlespeed).

As can be seen, all three cytometers were capable of scanningmultifunctional particles with varying levels of accuracy compared tothe static scans. Notably, the Guava instrument showed very goodreproducibility, but had rounded features, most likely due to a largelaser spot size (˜25 μm) compared to the dimension of each feature. TheAccuri showed fairly reproducible scanning but a significant amount ofnoise. The Partec showed considerable variability in scan intensity,likely due to a laser spot size that did not span the entire flowcell—most likely, particle brightness was dependent on where theparticle was positioned in the flow cell cross-section.

Nucleic Acid Detection We performed nucleic acid detection usingparticles with a single, wide fluorescent region to represent a“barcode” and a narrow probe region flanked by two inert regions. Wedetected microRNA let-7a spiked in at a level of 1 fmol into a 50 μlreaction with hybridization for 90 min at 55 C. Bound target was labeledwith streptavidin-phycoerythrin and particles were scanned using theMillipore Guava. The level of fluorescence in the probe region of theparticle indicated how much target was present in the assay. The resultsare shown in FIG. 23.

Again, the results were reproducible but showed rounding of signal atthe interfaces between various particle regions. For the highestsensitivity, our assay would benefit from green (532 nm) laserexcitation.

Example 11 Discrimination of Mature microRNA Targets from Precursors

According to the present invention, probes can be designed for labeling.This Example demonstrates detecting microRNAs using selectiveend-labeling to detect mature microRNA species without detection ofprecursor species.

We used a mature microRNA, the entire sequence of which is contained inone end of their precursor (3′ or 5′ depending on the exact microRNAspecies). If labeling is performed on the end common to both mature andprecursor, both species are labeled and quantified. To selectivelydetect mature species, labeling can be accomplished on the opposite endof the mature species, the end sequence which is contained internally onthe precursor. In the way, mature species can be detected withoutdetection of the precursor.

To demonstrate the detection of only mature microRNA species, syntheticmiR-143 mature and its precursor were used. The mature sequence formiR-143 appears on the 3′ end of the precursor. The sequences for thesespecies are given below in Table 6.

TABLE 6 Sequences for mature and precursor miR-143species. The mature sequence is underlined in the precursor sequence.miR-143 Species Sequence Mature 5′-UGAGAUGAAGCACUGUAGCUC-3′(SEQ ID NO: 20) Precursor 5′-GGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUGAAGCACUGUAGCUC-3′ (SEQ ID NO: 21)

Two batches of particles were used for this study—one contained miR-143probe designed for labeling the 3′ end of the target and the secondcontained a miR-143 probe designed for labeling the 5′ end of thetarget. The probe and adapter sequences used in this study are shown inthe tables below.

TABLE 7 Probe designs for labeling the 3′ or 5′ end of mature miR-143 species. The sequence for maturemiR-143 is underlined in each probe sequence,and the remaining sequence is designed tocapture the designated adapter for labeling. Probe (Target Label End)Sequence Probe 1  5′acryl-GATATATTTTAGAGCTACAGTGCTTC (3′ End):ATCTCA -3′ (SEQ ID NO: 22) Probe 2  5′acryl-GAGCTACAGTGCTTCATCTCAATTTA(5′ End): TATTT -3′ (SEQ ID NO: 23)

TABLE 8 Adapter sequences for 3′ and 5′ labeling. Adapter 1 has a 5′phosphate group and 3′ biotinylation, while Adapter 2 has a 5′biotinylation. Adapter (Target Label End)  Type Sequence Adapter 1  DNA5′phos-TAAAATATATAAAAAAAA (3′ End): AAAA-3′bio (SEQ ID NO: 24)Adapter 2  RNA 5′biotin-AAAAAAAAAUAUAAU  (5′ End): (SEQ ID NO: 25)

Probes 1 and 2 were designed to label bound target on the 3′ or 5′ endof mature miR-143, respectively. For 3′ labeling, a DNA adapter was usedwhile for 5′ labeling, an RNA adapter was used. These provided the mostefficient ligation for the designated end of the RNA target.

Particles were incubated, in a buffer containing 0.5M NaCl in TE, for 90minutes at 55 C with either 500 amols mature miR-143, 500 amols miR-143precursor, or no miR-143 target. After hybridization, particles werewashed in TE containing 0.05M NaCl. For particles bearing Probe #1, aligation was performed with T4 DNA ligase at concentration of 0.8 U/uland Adapter 1 at 40 nM. For particles bearing Probe #2, a ligation wasperformed with T4 RNA Ligase 2 at a concentration of 0.02 U/ul andAdapter 2 at 40 nM.

After 30 minute ligation at room temperature, particles were rinsed withTE containing 0.05M NaCl, and incubated with streptavidin-phycoerythrinreporter diluted to 2 ug/ml for 30 minutes at room temperature. Afterreporter conjugation, the particles were imaged using fluorescencemicroscopy. The signal-to-noise ratio, calculated as the average signaldivided by the standard deviation of the signal from the negativecontrol sample, was calculated for each miRNA species and labelingformat. The results are shown in the table below:

TABLE 9 Typical results from labeling experiment using 3′ and 5′labeling formats for mature and precursor miR-143 species. Thesignal-to-noise (SNR) represents the average fluorescence intensitysignal divided by the standard deviation of the negative control signal.Labeling Format miR-143 Species SNR Probe/Adapter #1 (3′) mature 75.9precursor 33.9 mature 21.6 Probe/Adapter #2 (5′) precursor ND

As can be seen, when Probe #1 is used with DNA ligase (Dnal) and Adapter#1, both mature and precursor miR-143 show detectable signal, althoughthe precursor is at a much lower level. When using RNA ligase 2 (Rnal2)with Adapter #2, mature miR-143 is the only target that is effectivelylabeled, while the precursor species is not detected (ND) above SNR=3.This shows effective discrimination for the detection of mature miRNAdetection over precursor species when labeling the 5′ end of the target.

Example 12 Two-Strip Encoding with Probe Functionalization

This example demonstrated that compositions described herein may besynthesized and functionalized for encoding, in particular, universalencoding.

Using stop-flow lithography as described in Patent U.S. Pat. No.7,709,554, the contents of which is incorporated herein by reference, weinitially synthesized rectangular particles bearing a stem-loop encodingprobe (SEQ ID NO:26) (/5Acryd/AATAAACACGGGAATAACCC, IDT, incorporated at10 uM), negative control region, probe anchor (SEQ ID NO:27)(/5Acryd/GATATATTTT, IDT, incorporated at 50 uM), and a second negativecontrol region. Particles were ˜120×60×35 um and each of the 4 stripswas ˜30 um thick. Particles were incubated with varying ratios offluorescently-labeled encoding adapter (SEQ ID NO:28)(5′-Phos-GTGTTTATAA-Cy3, IDT) to unlabeled adapter (SEQ ID NO:29)(5′-Phos-GTGTTTATAA-invdT, IDT). Each ligation mix contained NEBuffer #2with 250 nM ATP, 200U T4 DNA Ligase (all from New England Biosciences),and a total of 40 nM encoding adapters. Ligation was carried out for 30min at room temperature, with mixing at 1500 rpm on a thermomixer.Afterward, particles were rinsed 3× with TE buffer containing 50 mM NaCland 0.05% Tween-20. Particles were imaged on a Nikon Ti-S microscopeusing a 20× objective, NA=0.5, and a CCD Camera (Imaging Source). Scansof fluorescent intensity were plotted along the particle length and thefluorescent signals were measured and averaged for five particles ineach sample. Typical results are shown in FIG. 24.

Data demonstrates that the labeling worked, but the relationship offluorescence vs. adapter ratio was not linear. This implies a differencein hybridization or ligation rates between the fluorescent andnon-fluorescent adapters used. Unfortunately, the images at the 100%level were saturated, so it is difficult to use all 4 data points forcomparison. Raw and scaled data are shown in Table 10:

RAW DATA Sig SD COV Normalized Sig 100%  240.00 0.25 0.00 100%  1.00 50%201.63 2.07 0.01 50% 0.84 25% 140.43 3.55 0.03 25% 0.59 12.50%   92.072.45 0.03 12.50%   0.38

Furthermore, we used universal particles, synthesized using thestop-flow lithography process described above, bearing two encodingregions (with hairpin anchors) and a probe region (with linear anchor).Particles were ˜180 um long, 35 um wide, and ˜25 um thick with 4regions—UCode1 (synthesized at ˜10 uM), UCode2 (at ˜10 uM), inert, andUAnchor (at ˜50 uM). DNA sequences used in this study are as follows (asordered from Integrated DNA Technologies, 5-‘3’):

UCodel Probe = /5Acryd/AAT AAA CAC GGG AAT AAC  CC (SEQ ID NO: 30)UCode2 Probe = /5Acryd/AAT AAT GTG CCC AAT AAG  GG (SEQ ID NO: 31)UCode 1 Adapter Cy3 = /5Phos/GTG TTT ATT A/ 3Cy3Sp/ (SEQ ID NO: 32)UCode 1 Adapter invdT = /5Phos/GTG TTT ATT A/ 3InvdT/ (SEQ ID NO: 33)UCode 2 Adapter Cy3 = /5Phos/CAC ATT ATT A/ 3Cy3Sp/ (SEQ ID NO: 34)UCode 2 Adapter invdT = /5Phos/CAC ATT ATT A/ 3InvdT/ (SEQ ID NO: 35)

After particles were synthesized and rinsed, we prepared Ligation MasterMixes, each with 250 nM ATP (NEB), 200 U T4 DNA Ligase (NEB), 0.05%Tween-20 (Sigma), and DNA Adapter (given below) in a total of 500 ulNEBuffer #2 (NEB):

-   -   F1: 80 nM UCode Adapter 1 Cy3    -   N1: 80 nM UCode Adapter 1 invdT    -   F2: 80 nM UCode Adapter 2 Cy3    -   N2: 80 nM UCode Adapter 2 invdT

In a 96-well, 1.2 um filter-bottom plate (Millipore), we added mixes ofthe ligation mixtures as listed in Table 11.

F1 (ul) N1 (ul) F2 (ul) N2 (ul) W1: 1, 1 100 0 100 0 W2: 1, 0 100 0 0100 W3: 0, 1 0 100 100 0 W4: 1, 0.25 100 0 25 75 W5: 0.25, 1 25 75 100 0W6: 0.25, 0.25 25 75 25 75 W7: 0.25, 0 25 75 0 100 W8: 0, 0.25 0 100 2575

We then added 10 ul of particles to each well (˜200 particles) and putthe plate on mixer, and mixed at 1500 rpm for 30 min at room temp. Wethen used a filter unit to pull off excess buffer and rinse 2× with 200ul TE buffer with 0.05% Tween-20 (TET). For imaging, we added 60 ul ofTET to each well, mixed for 30 sec and then pipetted 35 ul from eachwell onto a glass slide. Each sample was sandwiched with an 18×18 mmcoverslip. We image particles with Nikon Ti-U microscope with ImagingSource CCD camera with brightness=30, gai=600, exposure=0.412 sec,gamma=150. After imaging 5 particles per sample, w used ImageJ to orientand crop images, and plugged data into Excel for analysis. The raw datafrom the analysis are shown in Table 12 below, the ratios representingthe amount of fluorescent adapter used (where 1=100%):

ratio 1 P1 SD1 COV ratio 2 P2 SD2 COV 1.00 85.71 2.26 0.03 1.00 78.861.98 0.03 1.00 83.45 3.85 0.05 0.00 2.55 0.64 0.25 0.00 1.42 0.25 0.181.00 77.96 4.22 0.05 1.00 85.32 3.55 0.04 0.25 46.14 1.25 0.03 0.2547.38 4.11 0.09 1.00 73.94 5.52 0.07 0.25 48.98 2.40 0.05 0.25 47.860.87 0.02 0.25 48.51 0.87 0.02 0.00 1.20 0.66 0.55 0.00 0.31 0.56 1.800.25 46.86 0.77 0.02

Shown below (FIG. 25a and b ) is a schematic of the particle design,sample fluorescent images from each ligation reaction, and average scansacross particles.

A plot of the measured fluorescence versus the adapter amount from eachligation mix are shown in FIG. 26, where “Code 1” and “Code 2” representthe average signal in the first and second code region, respectively.The encoding worked well. More importantly, the encoding was specific;the signal for each code region seemed to be independent of the other.As observed in the above experiments, the fluorescent level of each coderegion was not linear with respect to the amount of fluorescent adapter.The signals were very reproducible, especially at the 25% fluorescentadapter levels.

Example 13 Universal Encoding Using Template Functionalization

In this example, universal particles were made, bearing severalpolynucleotide templates for encoding.

As an example, particles were designed such that there were three activeregions separated by two inert regions, and they can be scanned by acommercial cytometer. The DNA templates with acrylate modification(denoted 5′ acry) used for encoding are listed below in Table 13:

Template name: Sequence UC1 5′acry-AATAAACACGGGAATAACCC-3′(SEQ ID NO: 36) UC2 5′acry-AATAATGTGCCCAATAAGGG-3′ (SEQ ID NO: 37) UC35′acry-AATAACTCTGGGAATAACCC-3′ (SEQ ID NO: 38)

These templates were used with particles of the design illustrated inFIG. 27. Hydrogel particles, consisting of poly(ethylene glycol), withthis design were made using flow lithography as discussed above. Theparticles were made with monomers containing the followingconcentrations of polynucleotide templates as listed in Table 14.

Barcode 1 Inert Probe Inert Barcode 2 UC1  50 uM NA NA NA NA UC2 2.5 uMNA 0.5 uM NA 2.5 uM UC3 NA NA NA NA  50 uM

For use in a flow cytometer, the UC2 template is functionalized with aCy5 modified adapter in order to trigger events in the RED2 channel. Forbarcoding, the UC1 and UC3 templates are functionalized with blends ofadapters (Cy3 modified, FAM-6 modified, or non-fluorescenct) in order toachieve distinct levels of fluorescence in the YEL channel of thecytometer for barcoding and distinct levels of fluorescence in the GRNchannel for orientation. The sequences of the adapters used are given inTable 15 below:

Adapter Name Sequence (5′-3′) UC1-A-Cy3 5′phos-GTGTTTATTA-Cy3 (SEQ ID NO: 39) UC1-A-NF 5′phos-GTGTTTATTA  (SEQ ID NO: 40) UC1-A-FAM65′phos-GTGTTTATTA-FAM6  (SEQ ID NO: 41) UC2-Cy5 5′phos-CACATTATTA-Cy5 (SEQ ID NO: 42) UC3-A-Cy3 5′phos-AGAGTTATTA-Cy3  (SEQ ID NO: 43)UC3-A-NF 5′phos-AGAGTTATTA  (SEQ ID NO: 44) UC3-A-FAM65′phos-AGAGTTATTA-FAM6  (SEQ ID NO: 45)

The number of distinguishable fluorescence levels in each barcode regiondepends on the accuracy of encoding, and performance characteristics ofthe cytometer being used. To determine the proper code dilutions tomaximize mutliplexing on a given flow cytometer, several blends offluorescent and non-fluorescent adapters may be tested for a givenencoding template. Several ratios of fluorescent to non-fluorescentadapters were explored by logarithmically varying the ratio betweenfluorescent and non-fluorescent and ligating multiple batches ofparticles a curve was generated as seen in FIG. 28. Templatefunctionalization via ligation with adapters was carried outsimultaneously for all templates for one hour at room temperature using0.8U T4 DNA ligase per ul, 40 nM total adapter for each encodingtemplate (fluorescent or non-fluorescent).

Several dilutions of UC1-A-Cy3 in UC1-A-NF were used to functionalizeuniversal particles in order to develop a titration curve for thefluorescence obtained. The curve in FIG. 28 shows the log(fluorescence)obtained using ratios of Cy3:NF adapter ranging from 0:1 to 1:1. Thiscurve was obtained using YEL fluorescence measurements from a GuavaeasyCyte 6HT.

Using this methodology, titration curves were made for the UC1 and UC3templates with Cy3 modified and non-fluorescent adapters. Typicalresults, showing log(fluorescence), are given in Table 16 below.

Barcode 1 Barcode 2 Corrected Corrected Ratio 1/Ratio Intensity COVRatio 1/Ratio Intensity COV 0 0 −0.79 17.40% 0 0 −0.8 10.80% 2560.0039063 −0.78 17.00% 256 0.00390625 −0.81 8.70% 128 0.0078125 −0.7420.40% 128 0.0078125 −0.78 11.60% 64 0.015625 −0.63 13.30% 64 0.015625−0.74 10.20% 32 0.03125 −0.52 12.60% 32 0.03125 −0.69 11.70% 16 0.0625−0.33 10.80% 16 0.0625 −0.6 10.00% 8 0.125 −0.1 10.00% 8 0.125 −0.449.00% 4 0.25 0.15 10.90% 4 0.25 −0.25 9.20% 2 0.5 0.43 10.90% 2 0.5 0.019.20% 1 1 0.69 12.90% 1 1 0.28 11.40%

Dilutions used for encoding were selected such that the expectedfluorescence levels had very little chance of overlap with an adjacentdilution, given the expected coefficient of variation (COV) in thesignals measured here. In order to obtain 5 levels for each barcoderegions, the following dilutions of non-fluorescent to Cy3-modifiedadapters in Table 17 were used:

Barcode 1 Adapter Log(intensity) NF:Cy3 −0.79 0 −0.42 21 −0.05 6.7 0.322.5 0.69 1

Barcode 2 Adapter Log(intensity) NF:Cy3 −0.80 0 −0.53 17.7 −0.26 4.10.01 2 0.28 1

With the possibility of generating 5 distinct levels of fluorescence ineach Barcode 1 and Barcode 2, a total of 25 unique combinations can beobtained. These dilutions were tested with the universal particlessynthesized in this Example. To differentiate the two coding regions, ahigher level of green (FAM-6) was added to the dilution series forBarcode 2. The fluorescent adapter for UC2 was also included in thefunctionalization to generate signal in RED2 which was used to triggerevents on the cytometer. Particles were functionalized via simultaneousligation with blends of adapters for UC1, UC2, and UC3 such that thetotal concentration of adapter for a given adapter was 40 nM. Reactionswere carried out at room temperature for 1 hour with 0.8 U/ul of T4 DNAligase present. Particles were rinsed in TE buffer and scanned using aGuava 6HT.

Example 14 Scanning Multi-Event Particles with Commercial Cytometers

In this example, universal particles made in Example 12 was used forscanning using commercial cytometers. A Millipore Guave easyCyte 6HT-2Las an exemplary cytometer can be used for scanning.

Here, particles were scanned on a cytometer using RED fluorescence totrigger events, yellow fluorescence to encode particles, and greenfluorescence to orient particles. As discussed, particles represented inFIG. 27 are comprised of three active regions (denoted Barcode 1, Probeand Barcode 2), separated by two inert regions. All three active regionscontain a Cy5-modified nucleic acid to trigger events in the RED2channel of a Guava easyCyte cytometer. The level of Cy5 in the proberegion was intentionally made to be approximately one half that in thebarcoding regions. The two barcode regions contain varying levels ofCy3-modified oligonucleotides. The levels of Cy3 in each barcode region,detected in the YEL channel of the Guava cytometer, are used to give theparticle a unique encoding signature. In addition, a FAM6-modifiedoligonucleotide is incorporated in the barcoding regions, with a higherlevel in Barocde 1, in order to provide a means of orientation. Amixture containing 25 different particle barcodes, with 5 unique levelsof Cy3 fluorescence in Barcode 1 and 5 unique levels in Barocde 2, wereused to demonstrate proof-of-concept.

A threshold of 500 set on the RED2 channel with the Guava 6HT wassufficient to allow identification of all three regions of the particle.Hundreds of particles, at a concentration of approximately 20 permicroliter in TE buffer, were scanned at 0.6 microliters per second. Theevents associated with the particles, plotted on YEL (barcoding color)versus RED2 (trigger color) are shown in FIG. 29, along with YEL versusGRN (for orientation). The probe region of the particle appears in thelower left hand side of the plot, with lower levels of green (FAM-6™)and yellow (Cy3™). The two coding regions of the particles show up asbands on the upper right hand corner of the plot. A total of ten bandscan be discerned on the plot, comprising of five codes on the Barcode 1region of the particle and five codes on the Barcode 2 region. The rawvalues represented on these plots are then exported into a FCS file forfurther analysis. All events exported in the CSV are store in temporalsequence.

Custom software was used to analyze the events exported from the Guavasoftware and reconstruct them, based on patterns in the RED2 and GRNfluorescence. The software sorts through the sequence of events toassess whether three subsequent events fit the expected patterns forRED2 and GRN fluorescence. If the pattern is fit, the events are groupedas a particle and can be analyzed for barcode in YEL fluorescence andoriented by GRN fluorescence. After reconstruction, a more coherent plotcan be composed using the level of yellow intensity (Cy3™) on Barcode 1vs. that of Barcode 2 (designated code 1 and code2, respectively). Thisplot is shown in FIG. 30. Ellipses are used to identify clusters ofparticles that are associated with each of the 25 barcodes present. Ascan be seen, the five levels of fluorescence in Barcode 1 (code 1) andBarcode 2 (code 2) can be readily distinguished.

In addition to determining the barcode, the custom software alsoquantifies the fluorescence associated with captured target in the proberegion of the particle, the information of which is stored as the secondof the three events associated with a particle. When using a reportingfluorophore that can be detected in the YEL channel, the level of YELfluorescence in this region indicates the quantity of target present.

Example 15 Development of One-Spot Isothermal Nucleic Acid AmplificationAssays

This example further illustrates using encoded particles in accordancewith the present invention in various applications, such as nucleic acidamplification assays. As previously demonstrated, we has developedvarious compositions and methods, providing (1) sub-attomolesensitivity, (2) single-nucleotide specificity, (3) rapid scanning, (4)a virtually unlimited encoding density, and (5) low cost. For example,the high performance of our assay is shown for microRNA targets in aboveExamples, and FIG. 31. The simplicity of our particle synthesis, one-potassay, and single-color detection described herein enables a new classof low-cost diagnostic tools.

In this project, we will use encoded hydrogel particle assay to developa point-of-care system that (1) can perform accurate panel-based testson DNA or RNA from >10 pathogens at once, (2) uses a one-pot, isothermalassay that is rapid and easy to use, and (3) utilizes low-costdisposable cartridges in a hand-held device. We are developing one-potassays in which we amplify specific genomic targets of pathogens,hybridize the amplicons to barcoded gel particles, and quantify thebound amplicons in a single closed tube, with a single user intervention(sample loading). Multiple species-specific targets will be amplifiedusing isothermal, helicase-dependent amplification (HDA).Fluorescently-labeled amplicons will be free to diffuse into the encodedhydrogel particles and hybridize to their complementary nucleic acidprobes embedded throughout (FIG. 32). The flexibility of our innovativemicrofabrication process allows us to precisely tune the pore size orparticles to exclude helicase enzymes (˜4.5 nm), which would unwindbound targets. Due to this advantage, the whole process can be carriedout without user intervention. After <1 hour, particles will be scannedrapidly in a flow-through channel using fluorescence to read the barcodeof each particle and quantify the corresponding targets. It is ourintention to make the system cartridge-based with disposable units thatcan be interfaced with a portable analysis unit.

We further developed one-pot assays as described in various embodimentsabove, using standard PCR and has recently begun to investigateisothermal assays for the purpose of this project. We used λ-phage DNAas a model system for assay development. First, we designed Tm-matchedprimers against 2 target regions of lambda with a cross-check againsthuman genomic DNA to avoid non-specific amplification. The ampliconswere designed to be ˜60 bp in length. Probes were designed to targeteach amplicon, containing the complementary sequence excluding thebinding site for the forward primer. We performed one-pot assays usingboth standard PCR and isothermal amplification (FIG. 33).

For each assay, we prepared PCR mixes containing a single primer set(forward primer labeled with Cy3), ˜50 encoded gel particles with twospatially-separated probes regions for the amplicons, and either λ-phageDNA or human genomic DNA. Using both standard PCR and isothermalamplification, we were able to show specific amplification andhybridization for each amplicon generated and no non-specificamplification of human genomic DNA. We performed a serial dilution ofλ-phage from 11,000-11 copies per reaction. Using primer set #1, we wereable to detect ˜11 copies of template in our preliminary studies using aone-pot assay with standard PCR. Although sensitivity has not beenassessed for the isothermal reaction, the signals observed on particlesafter 60-min reaction were stronger than those obtained from standardPCR after 40 cycles.

Design of Amplification Primers and DNA Detection Probes

For any pathogen, it is necessary to identify genomic targets that areboth specific to the pathogen, and conserved over strains. We will buildon the work of others developing PCR-based assays for the four pathogensof interest. Targets for genomic HIV RNA include: the pol-integraseregion and the env and gag genes. Targets used for PCR-basedidentification of for typhoid bacterium genome include the tyv, flag,viaB, and ratA genes. Conserved regions for the malarial parasite genomeinclude the 18 s rRNA gene and the circumsporozoite (CS) gene. Fordengue virus, Gurukumar et al. targeted a conserved region in the 3′UTRof the viral genome. Initially, our experiments are designed to targetsimilar regions for these pathogens.

For multiplexed isothermal amplification, it is necessary to designcompatible primer sets that (1) have similar melting temperatures, (2)do not form hetero-dimers, and (3) specifically and efficiently amplifythe targets identified for each pathogen species. Because we aredeveloping a “one-pot” assay where the particles are present in theamplification reaction, we have additional considerations including (1)avoiding 3′-extension of the DNA probes embedded in the particleprobe-regions, and (2) keeping amplicons small (<100 bp) for rapiddiffusion into our particles where they will hybridize. In approachingthis challenge, we will learn from an extensive body of literature forprimer design in multiplexed amplification.

As shown in FIG. 34, primers will be designed to have meltingtemperatures near 55° C., be ˜20 bp in length, and provide amplicons ˜60bp in size. The forward primers will contain a single Cy3 label forfluorescence detection. For each of the pathogen species, we will designseveral sets of primers that meet the aforementioned requirements.Primer design will be accomplished as follows:

First, potential primers sets will be identified for the species ofinterest (dengue, typhoid, malaria, and HIV as well as λ-phage and MS2controls) for commonly-targeted, conserved genomic regions using aprimer-design program like Primer3.

Second, each potential primer identified will be assessed forspecies-specificity via BLAST search.

Third, a script will be written in MATLAB to assess dimer-formation withall other primers (using nearest neighbor calculations), and to identifya total of 30 primer sets (5 for each of the four pathogens and twocontrols) that meet all requirements.

Optimization of Helicase-Dependent Amplification (HDA) for DNADetection.

To maximize the probability of success in developing a workingisothermal amplification technique, we will begin with commerciallyavailable kits and standard protocols, using k-phage as a model system.We will use the IsoAmp® kit (New England Biosciences) to performisothermal amplification on ˜5000 copies of λ-phage spiked into humangenomic DNA as a model system. We will optimize several parametersincluding (1) primer concentrations (from 0.1 μl-10 μM), (2) primerlength (from 20-26 bp), (3) amplification temperature (from 50-65 C),and (4) reaction time (from 10-120 min). The efficiency and yield of theisothermal reaction will be assessed and compared to the yield of astandard 30-cycle PCR reaction that utilizes the same primers and targetregions. Polyacrylamide Gel Electrophoresis (PAGE) will be used to makethis qualitative comparison, with target band intensity as thestandardized metric.

After optimizing reaction conditions, the primer sets for the other DNAspecies (P. falciparum, and S. tyhpi) will be interrogated forefficiency and specificity. Again, we will assess amplificationefficiency for each primer set by quantifying the amount of targetproduced in 10, 30, and 90 min isothermal amplification (via PAGE).Specificity will be assessed by performing PCR with a primer set for agiven species using human genomic DNA spiked with ˜5000 copies ofgenomic species for all other species. Specific robust reactions willshow amplification of only the target sequence. Of the 5 primer setsdesigned for each species, we will use the three most efficient setsthat show good specificity.

The three primer sets for each species will be used in a multiplexedamplification assay with one target present at a time. For multiplexedreactions, target amplification will be accomplished using a fluorescentforward primer, as shown in FIG. 34. For each reaction, theamplification product will be quantified using a 30 min incubation withbarcoded gel particles bearing probes for each amplicon (FIG. 35). TheDNA probes for each particle will be designed to span the reverse primerand internal region, and will be 3′ capped to avoid extension as shownin FIG. 35. This design will allow for one-pot amplification/capture insubsequent studies.

Ideally, the fluorescent signals observed on the particles would beconsistent over the 3 amplicons generated for each species. Ifsignificant differences in amplification/capture efficiency are observedfor the multiplexed amplification, several reaction conditions will bevaried in order to normalize the amount of amplicon captured on eachparticle probe region. First, the relative amounts of primers can beadjusted accordingly to alter the reaction kinetics. Second, primerlength can be adjusted in order to change binding efficiency—this willlikely affect the primer Tm and increase nonspecific amplification, andis therefore not desirable. Third, we have demonstrated that the rate ofcapture can be adjusted in a very predictable manner by changing theconcentration of probe in each region of the particles.

After normalizing quantified signal for each species, we will performone-pot assays where amplification and hybridization are completed inthe same reaction. We will determine the effects that the particles haveon the sensitivity and specificity of the primer sets. Iterativeoptimization of primer and probe sequences may be necessary, along withreaction temperature and duration. In the case of multiplexed, one-potassays, we will image particles in both static (microscopy) andflow-through modes. We will monitor and compare sensitivity andreproducibility of the two approaches—these will be importantconsiderations when designing the integrated system proposed in Example17.

Reverse Transcription of Pathogen Genomic Material.

While the genomic DNA of P. falciparum and S. typhi can be directlyamplified, the detection of HIV-1 and dengue virus, both ssRNA viruses,will require reverse transcription of genomic RNA to cDNA foramplification and analysis. This requires the addition of a reversetranscriptase enzyme into the isothermal amplification reaction. Reversetranscription has been successfully coupled with Helicase-DependentAmplification, and isothermal RT-HAD kits are available commercially(IsoAmp®, NE Biolabs). This is the same kit being used in the previousstudies.

We will start with a standard recommended protocol for RNA reversetranscription and cDNA amplification, using Phage MS2 as a model systemfor optimization. Using the 5 primer sets originally identified forPhage MS2, we will perform a similar optimization as done for DNAamplification. Once optimized, we will assess primer sets for thepathogen RNA targets, again quantifying amplification efficiency andspecificity. Using the 3 best primer sets for each RNA species, we willperform a multiplex amplification for each. Again, amplicons will bequantified using encoded gel particles in both static and flow-throughmodes.

Optimization of One-Pot Assay for Multiplexed Pathogen DNA or RNADetection.

Having independently optimized both multiplexed detection of DNA targetsand RNA targets, we will combine these assays, and optimize forperformance and speed. Using a human genomic DNA background, we willspike genomic material from each pathogen into samples at concentrationsranging from 1-100,000 copies. We will investigate and optimize primerconcentrations, enzyme concentration, assay duration, and assaytemperature. We will evaluate the performance of the assay for eachpathogen, measuring specificity, limit of detection, and sensitivity at100 copies/rxn. It is our goal to demonstrate 95% sensitivity for allpathogens at 100 copies/rxn with an assay time of 60 min.

Although the use of isothermal amplification with a one-stepamplification/hybridization reaction capable of detecting both DNA andRNA species in a single sample is ideal, there are several alternativeapproaches which are perhaps less attractive, but more likely forsuccess.

For example, if Helicase-Dependent Amplification (HDA) does not proveeffective, several other isothermal methods will be investigatedincluding Loop-Mediated Isothermal Amplification (LAMP),Strand-Displacement Amplification (SDA), and Nucleic Acid Sequence-BasedAmplification (NASBA). Importantly, a NASBA-based assay has previouslybeen approved by the FDA for the detection of HIV-1 and so would serveas an obvious next choice for RNA detection. Alternatively, standard PCRmay be used. In fact, microfluidic methods for PCR amplification arebecoming very common so the use of this technique would not be out ofthe question. Also, if the detection of RNA pathogens (which requiredreverse transcription) and DNA pathogens in the same tube gives rise toinsurmountable complications, these assays can be separated into twodistinct tests.

In some embodiments, as an alternative approach to one-pot assays,two-step amplification/hybridization can be use in accordance with thepresent invention. If the particles interfere in any way with theamplification process, it may be necessary to perform amplification andhybridization separately. Envisioning a cartridge-based system in whichthis technology can be implemented, this assay can still be accomplishedon-chip but will require slightly more sophisticated liquid handling.Although this is not the ideal situation, it is manageable and canfeasibly meet the needs of diagnostics in the developing world.

Example 16 Validation of One-Pot Assay for Multiplexed PathogenDetection

After developing a one-pot assay for the multiplexed detection ofpathogens in Example 15, we will validate it using clinically-relevantsamples and benchmark it against pathogen-specific assays developed forquantitative PCR, the current gold standard for nucleic-acid basedpathogen diagnostics. This objective will be important in demonstratingthe clinical utility of this assay.

We will obtain a representative set of clinically-relevant samples fromseveral collaborators. Without being bound to any particular theory, itis believed that the samples we obtain will be well-preserved. This isespecially important for RNA detection as RNA is rapidly degraded byRNase activity. If the available sample volume permits, we will performquality control via DNA/RNA sizing with an Agilent Bioanalyzer. Anotherassumption is that these samples will be representative of the samplesthat would be obtained in the field when our technology is deployed.Ideally, the samples would span a broad range of pathogen load, andstates of patients' immunologic response.

There are several stages in the validation of our assay. Initially, wewill investigate various methods for purifying nucleic acids from wholeblood and determine compatibility with our assay for each pathogen. Thiswill be important in determining which purification technologies couldbe integrated with our platform after this initial research project iscompleted. We will ideally be able to select one isolation techniquethat performs well for all pathogens, and use it for all validationtests. We will purify nucleic acids from the clinical samples (blood orplasma) provided by our collaborators and test the samples using ourone-pot test and also commercially-available pathogen qPCR kits. Thiswill allow a direct benchmark of our assay against the currentstate-of-the art. Details for each part of the validation process aregiven below.

Assessment of nucleic acid purification techniques. There are severalmethods for extracting nucleic acids from whole blood, plasma, or serum.Most of the kits are specific for either RNA or DNA, though a few kitscan be used to extract both. We will investigate severalcommercially-available kits including:

DNA Extraction: QIAamp Blood DNA Mini Extraction Kit (QIAGEN), GenomicDNA Extraction Kit (Bioneer), Extract-N-Amp Blood PCR Kits (Sigma).

RNA Extraction: QIAmp Viral RNA Mini Extraction Kit (QIAGEN), Viral RNAExtraction Kit (Bioneer).

Simultaneous Extraction of DNA and RNA: QIAamp MinElute Virus Spin Kit,QIAamp UltraSens Virus Kit, NucleoSpin Virus Kit (Macherey-Nagel).

Clearly, the optimal mode for multiplexed assays is the use of a singleextraction method for parallel isolation of pathogen DNA and RNA. Wewill devote a significant amount of effort into identifying andoptimizing a method for dual nucleic acid extraction that functions wellwith our one-pot assay. To assess compatibility, we will usewell-characterized clinical samples containing each pathogen and performextraction with each of the kits. The samples will subsequently beassessed with our one-pot assay and also validated using qPCR kitsspecifically designed for each pathogen.

Assay on Clinical Samples with Direct Comparison to qPCR. Nucleic acidsfrom clinical samples (at least 30 for each pathogen type) will bepurified using the optimal method determined in the previous section. Wewill perform a one-pot, multiplexed assay for the detection of pathogensin each sample and compare our results to qPCR assays specificallydesigned for each pathogen. For three of the four pathogens beinginvestigated, there are several qPCR kits available. At the time wereach this objective, we will select the kit that has shown bestperformance and has received certification for diagnostic testing:

-   Dengue: Primer Design, Ltd. and Genome Diagnostics-   Malaria: Primer Design, Ltd., AccuPower, and Genome Diagnostics-   HIV-1: Primer Design, Ltd., and Genome Diagnostics-   Typhoid: To our knowledge, there is no commercially-available qPCR    assay for S. tyhpi. There is a multiplex PCR-based approach by Kumar    et al. that will be used in place of qPCR if no test has been    developed by the time we reach this objective of the project.

For relative comparison of sensitivity, we will also make serialdilutions of a representative sample for each pathogen type and analyzethem using both our assay and the qPCR standard. A strong correlation ofour assay results with the state-of-the art is important for validation.If our assay performs less desirably than expected, we will troubleshootthe assay by re-evaluating the regions targeted, primer design, andassay conditions. We will work closely with our collaborators forguidance in resolving any issues.

Example 17 Development of a Proof-of-Concept Integrated System

After successfully developing an assay, it is important to beginconceptualizing methods for the assay to be implemented on chip. Forthis reason, we will explore methods for performing one-pot assay andanalyzing particles in a single chamber. This will require thedevelopment of an integrated system capable of precise temperaturecontrol with capabilities for fluorescence imaging for static particleanalysis or rapid signal acquisition for flow-through analysis. Thissystem will allow periodic analysis of the particles to assess theprogress of reaction. As a significant improvement over end-pointanalysis, we believe that this method of analysis can be calibrated toprovide precise quantitative analysis of pathogen load. In this Example,we aim to develop an integrated system to perform rapid, one-pot assayswith the ability to accurately quantify pathogen nucleic acids.

As the simplest initial approach, we will use a commercially-availabletemperature-controlled cell perfusion chamber with static imaging on amicroscope. We will perform several studies to evaluate the use of aone-pot chamber reaction for pathogen detection and also assess thefeasibility of performing quantitative analysis with periodic imageanalysis. After successful implementation, we will integrate the heatedflow chamber into a stand alone device with an LED illumination sourceand a CCD camera to acquire images. This represents an important steptoward developing a cartridge-based system that would ultimately bedeployed in developing countries. More details on the specificactivities for this objective are given below.

One potassays in a heated flow cell. We will use acommercially-available heated flow cell, similar to those sold byBioptechs. These flow cells feature (1) customizable channel design, (2)multiple interfaces for sample introduction, (3) precise temperaturecontrol with +/−0.2° C. stability, and (4) a standard design formounting on any microscope. Initially, we will utilize a simplerectangular flow chamber for assay and analysis. We will premix thereaction mixture to include the sample of interest, isothermalamplification reagents, and ˜50 particles for each of the four pathogensand two controls. The device will be pre-heated to the isothermalamplification temperature (˜55° C.) and the reaction mixture will beintroduced into the reaction chamber. Using a standard invertedmicroscope with a 5× objective (for large field of view), singleexcitation color, and single detection color, particles will be imagedthroughout the course of amplification, likely every 5 minutes. Eachimage will be analyzed to estimate the amount each amplicon generated,based on probe-region fluorescence. After 60 min reaction, this dynamicdata will be used to estimate the amount of template initially present.For a proof-of-concept, we will use the two controls, k-phage DNA andPhage MS2, in order to characterize system performance and ability toprovide quantitative data.

Design and construction of an integrated assay/scanning system. Aftersuccessful implementation of a microscope-based system, we willintegrate the flow cell into a custom optical system. We will utilize ahomogeneous LED illumination, a low-magnification lens, and a CCD chip.The LED array, CCD, and heated flow cell will be interfaced with alaptop computer for control, image acquisition, and analysis. The unitwill be thoroughly tested, and results will be compared to thoseobtained previously in this project. We will re-evaluate the sensitivityand specificity of detection for each pathogen using this setup. We willalso investigate the quantitative dynamic range of the system by spikingin targets from 1-1M copies. We take measures to ensure that performanceis not compromised in an integrated system.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations and web pages, regardless of the format of such literatureand similar materials, are expressly incorporated by reference in theirentirety. In the event that one or more of the incorporated literatureand similar materials differs from or contradicts this application,including defined terms, term usage, described techniques, or the like,this application controls.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

Other Embodiments and Equivalents

While the present disclosures have been described in conjunction withvarious embodiments and examples, it is not intended that they belimited to such embodiments or examples. On the contrary, thedisclosures encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.Accordingly, the descriptions, methods and diagrams of should not beread as limited to the described order of elements unless stated to thateffect.

Although this disclosure has described and illustrated certainembodiments, it is to be understood that the disclosure is notrestricted to those particular embodiments. Rather, the disclosureincludes all embodiments that are functional and/or equivalents of thespecific embodiments and features that have been described andillustrated.

We claim: 1-30. (canceled)
 31. A method for detecting the presenceand/or abundance of target nucleic acids in a sample, comprising stepsof: a) contacting a plurality of nucleic acid probes with a samplecomprising target nucleic acids, wherein each nucleic acid probecomprises a capturing sequence for binding an individual target nucleicacid and an adjacent adapter sequence for binding an individualuniversal adapter and further wherein the plurality of nucleic acidprobes are attached to a particle comprising two or more encodingregions and one or more probe regions that are distinct from theencoding regions, wherein each encoding region is separated from eachprobe region or encoding region by an inert region, wherein theplurality of nucleic acid probes are associated with the one or moreprobe regions and wherein the two or more encoding regions beardetectable moieties that give identity of the plurality of nucleic acidprobes; b) providing one or more universal adapters under conditionsthat permit binding of the individual universal adapter to an individualnucleic acid probe hybridized with the individual target nucleic acid,wherein the one or more universal adapters are labeled with detectablesignals; c) coupling the individual universal adapter to the individualtarget nucleic acid hybridized to the same individual nucleic acid probewith an enzyme; d) removing uncoupled universal adapters under stringentconditions; and e) detecting the presence of the detectable signalsassociated with the one or more universal adapters coupled to the targetnucleic acids hybridized to the plurality of nucleic acid probes and thedetectable moieties associated with the one or more encoding regionsthat give the identity, thereby detecting the presence and/or abundanceof the target nucleic acids in the sample.
 32. The method of claim 31,wherein the individual target nucleic acid and the individual universaladapter bind to the same individual nucleic acid probe at the same timeor sequentially.
 33. The method of claim 31, wherein the detecting instep (e) comprises scanning the particle using a flow-through device.34. The method of claim 33, wherein the scanning is performed above themelting temperature of the universal adapter but below the meltingtemperature of the coupled target-adapter.
 35. The method of claim 33,wherein the flow-through device is a flow cytometer.
 36. The method ofclaim 31, wherein the method further comprises a step of quantifying theamount of the target nucleic acids.
 37. The method of claim 31, whereinthe target nucleic acids comprise microRNA.
 38. The method of claim 37,wherein the adapter sequence on the probe is positioned at the end ofthe capturing sequence in such a manner that allows labeling of a maturemicroRNA species but not precursor microRNA species.
 39. The method ofclaim 31, wherein the target nucleic acids comprise multiple species ofnucleic acids and wherein the multiple species of nucleic acids containvariable nucleotide sequence at one end and identical nucleotidesequence at the other end.
 40. The method of claim 31, wherein thecapturing sequence comprises at least 10 bases.
 41. The method of claim31, wherein the adapter sequence comprises 10-20 nucleotides.
 42. Themethod of claim 31, wherein the individual nucleic acid probe comprises10-50 nucleotides.
 43. The method of claim 31, wherein the particle is ahydrogel particle.
 44. A method for detecting the presence and/orabundance of target nucleic acids in a sample, comprising steps of: a)incubating a plurality of particles with a sample comprising targetnucleic acids, wherein each of the plurality of particles comprises twoor more encoding regions and one or more probe regions that are distinctfrom the encoding regions, wherein each encoding region is separatedfrom each probe region or encoding region by an inert region, andwherein the one or more probe regions are embedded with multiple nucleicacid probes and wherein the two or more encoding regions bear detectablemoieties that give identity of the multiple nucleic acid probes andfurther wherein each nucleic acid probe comprises a capturing sequencefor binding a target nucleic acid and an adjacent adapter sequence forbinding a universal adapter; b) providing one or more universal adaptersunder conditions that permit binding of an individual universal adapterto an individual nucleic acid probe hybridized with an individual targetnucleic acid, wherein the one or more universal adapters are labeledwith detectable signals; c) coupling the individual universal adapter tothe individual target nucleic acid hybridized to the same individualnucleic acid probe with an enzyme; d) removing uncoupled universaladapters under stringent conditions; and e) scanning the plurality ofparticles by a flow-through device to detect the presence of thedetectable signals associated with the one or more universal adapterscoupled to the target nucleic acids hybridized to the nucleic acidprobes and the detectable moieties associated with the one or moreencoding regions that give the identity, thereby detecting the presenceand/or abundance of the target nucleic acids in the sample.
 45. Themethod of claim 44, wherein the target nucleic acids are microRNAs. 46.The method of claim 44, wherein the adapter sequence on the probe ispositioned at the end of the capturing sequence in such a manner thatallows labeling of a mature microRNA species but not precursor microRNAspecies.
 47. The method of claim 44, wherein the particle is a hydrogelparticle.
 48. The method of claim 44, wherein the detectable signals arefluorescent signals.
 49. The method of claim 44, wherein the capturingsequence comprises at least 10 bases.
 50. The method of claim 44,wherein the adapter sequence comprises 10-20 nucleotides.
 51. The methodof claim 44, wherein the individual nucleic acid probe comprises 10-50nucleotides.
 52. The method of claim 44, wherein the flow-through deviceis a flow cytometer.
 53. A method for detecting the presence and/orabundance of target microRNA in a sample, comprising steps of: a)contacting a plurality of DNA probes with a sample comprising targetmicroRNAs, wherein each DNA probe comprises a capturing sequence forbinding an individual target microRNA and an adjacent adapter sequencefor binding an individual universal adapter and further wherein theplurality of DNA probes are attached to a particle comprising two ormore encoding regions and one or more probe regions that are distinctfrom the encoding regions, wherein each encoding region is separatedfrom each probe region or encoding region by an inert region; andwherein the plurality of DNA probes are associated with the one or moreprobe regions and the two or more encoding regions bear detectablemoieties that give identity of the plurality of DNA probes; b) providingone or more universal adapters under conditions that permit binding ofan individual universal adapter to the individual DNA probe hybridizedwith the individual target microRNA, wherein the one or more universaladapters are labeled with detectable signals; c) coupling the individualuniversal adapter to the individual target microRNA hybridized to thesame individual DNA probe with an enzyme; d) removing uncoupleduniversal adapters under stringent conditions; and e) detecting thepresence of the detectable signals associated with the one or moreuniversal adapters coupled to the target microRNAs hybridized to the DNAprobes and the detectable moieties associated with the one or moreencoding regions that give the identity, thereby detecting the presenceand/or abundance of the target microRNAs in the sample.